Numerical Simulation of Passage of a Neutrophil through a Rectangular Channel with a Moderate Constriction

The authors have previously presented a mathematical model to predict transit time of a neutrophil through an alveolar capillary segment which was modeled as an axisymmetric arc-shaped constriction settled in a cylindrical straight pipe to investigate the influence of entrance curvature of a capillary on passage of the cell. The axially asymmetric cross section of a capillary also influences the transit time because it requires three-dimensional deformation of a cell when it passes through the capillary and could lead to plasma leakage between the cell surface and the capillary wall. In this study, a rectangular channel was introduced, the side walls of which were moderately constricted, as a representative of axially asymmetric capillaries. Dependence of transit time of a neutrophil passing through the constriction on the constriction geometry, i.e., channel height, throat width and curvature radius of the constriction, was numerically investigated, the transit time being compared with that through the axisymmetric model. It was found that the transit time is dominated by the throat hydraulic diameter and curvature radius of the constriction and that the throat aspect ratio little affects the transit time with a certain limitation, indicating that if an appropriate curvature radius is chosen, such a rectangular channel model can be substituted for an axisymmetric capillary model having the same throat hydraulic diameter in terms of the transit time by choosing an appropriate curvature radius. Thus, microchannels fabricated by the photolithography technique, whose cross section is generally rectangular, are expected to be applicable to in vitro model experiments of neutrophil retention and passage in the alveolar capillaries.     

Computational modeling of RBC and neutrophil transit through the pulmonary capillaries.

A computational model of the pulmonary microcirculation is developed and used to examine blood flow from arteriole to venule through a realistically complex alveolar capillary bed. Distributions of flow, hematocrit, and pressure are presented, showing the existence of preferential pathways through the system and of large segment-to-segment differences in all parameters, confirming and extending previous work. Red blood cell (RBC) and neutrophil transit are also analyzed, the latter drawing from previous studies of leukocyte aspiration into micropipettes. Transit time distributions are in good agreement with in vivo experiments, in particular showing that neutrophils are dramatically slowed relative to the flow of RBCs because of the need to contract and elongate to fit through narrower capillaries. Predicted neutrophil transit times depend on how the effective capillary diameter is defined. Transient blockage by a neutrophil can increase the local pressure drop across a segment by 100--300%, leading to temporal variations in flow and pressure as seen by videomicroscopy. All of these effects are modulated by changes in transpulmonary pressure and arteriolar pressure, although RBCs, neutrophils, and rigid microspheres all behave differently.     

Distribution of pulmonary capillary transit times in recruited networks

When pulmonary blood flow is elevated, hypoxemia can occur in the fastest-moving erythrocytes if their transit times through the capillaries fall below the minimum time for complete oxygenation. This desaturation is more likely to occur if the distribution of capillary transit times about the mean is large. Increasing cardiac output is known to decrease mean pulmonary capillary transit time, but the effect on the distribution of transit times has not been reported. We measured the mean and variance of transit times in single pulmonary capillary networks in the dependent lung of anesthetized dogs by in vivo videofluorescence microscopy of a fluorescein dye bolus passing from an arteriole to a venule. When cardiac output increased from 2.9 to 9.9 l/min, mean capillary transit time decreased from 2.0 to 0.8 s. Because transit time variance decreased proportionately (relative dispersion remained constant), increasing cardiac output did not alter the heterogeneity of local capillary transit times in the lower lung where the capillary bed was nearly fully recruited.     

Physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries

Although the lung is known to be a major site of neutrophil margination, the anatomic location of these sequestered cells within the lung is controversial. To determine the site of margination and the kinetics of neutrophil transit through the pulmonary microvasculature, we infused fluorescein isothiocyanate-labeled canine neutrophils into the pulmonary arteries of 10 anesthetized normal dogs and made fluorescence videomicroscopic observations of the subpleural pulmonary microcirculation through a window inserted into the chest wall. The site of fluorescent neutrophil sequestration was exclusively in the pulmonary capillaries with a total of 951 labeled cells impeded in the capillary bed for a minimum of 2 s. No cells were delayed in the arterioles or venules. Transit times of individual neutrophils varied over a wide range from less than 2 s to greater than 20 min with an exponential distribution skewed toward rapid transit times. These observations indicate that neutrophil margination occurs in the pulmonary capillaries with neutrophils impeded for variable periods of time on each pass through the lung. The resulting wide distribution of transit times may determine the dynamic equilibrium between circulating and marginated neutrophils.     

Shear-induced resistance to neutrophil activation via the formyl peptide receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and α(M)β(2) integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1-4.0 dyn/cm(2)) in a cone-and-plate viscometer for 1-120 min showed a significant reduction in both α(M)β(2) integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and α(M)β(2) integrin activation in the presence of soluble ligand.     

Effect of steady and oscillatory shear stress on F-actin content and distribution in neutrophils

We investigated neutrophil activation, specifically F-actin content and distribution, in situations mimicking the in vivo environment using steady and oscillatory shear. Under low steady shear (<150 s(-1)) F-actin levels were decreased for both treated (n-formyl-L-methioryl-L-leucyl-L-phenylalanine (fMLP)) and untreated neutrophils. The F-actin content increased with a change to higher steady shear levels. Neutrophils show the same behavior of decreased F-actin content for oscillatory shear (26.7 s(-1)) as they did for steady shear. In both situations, the low shear levels caused a decrease in F-actin content. However, as the magnitude of the shear rate increased, cells showed a reversal to increasing F-actin content. Shear caused a decrease in F-actin in the cell cortex for both control and fMLP treated cells. Ctyochalasin B (CB), a common F-actin assembly blocker, significantly decreased F-actin content. The results indicate that neutrophils regulate their actin network based on the level and type of shear stress they encounter in the bloodstream.     

Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis

Surfactant protein A (SP-A), a pulmonary lectin, plays an important role in regulating innate immune cell function. Besides accelerating pathogen clearance by pulmonary phagocytes, SP-A also stimulates alveolar macrophage chemotaxis and directed actin polymerization. We hypothesized that SP-A would also stimulate neutrophil chemotaxis. With the use of a Boyden chamber assay, we found that SP-A (0.5-25 microg/ml) did not stimulate chemotaxis of rat peripheral neutrophils or inflammatory bronchoalveolar lavage (BAL) neutrophils isolated from LPS-treated lungs. However, SP-A affected neutrophil chemotaxis toward the bacterial peptide formyl-met-leu-phe (fMLP). Surprisingly, the effect was different for the two neutrophil populations: SP-A reduced peripheral neutrophil chemotaxis toward fMLP (49 +/- 5% fMLP alone) and enhanced inflammatory BAL neutrophil chemotaxis (277 +/- 48% fMLP alone). This differential effect was not seen for the homologous proteins mannose binding lectin and complement protein 1q but was recapitulated by type IV collagen. SP-A bound both neutrophil populations comparably and did not alter formyl peptide binding. These data support a role for SP-A in regulating neutrophil migration in pulmonary tissue.     

Alteration of red cell membrane viscoelasticity by heat treatment: effect on cell deformability and suspension viscosity

The membrane shear elastic modulus (mu) and the time constant for extensional shape recovery (tc) were measured for normal, control human red blood cells (RBC) and for RBC heat treated (HT) at 48 degrees C. Three separate methods for the measurement of mu were compared (two used a micropipette and one employed a flow channel), and the membrane viscosity (n) was calculated from the relation n = mu. tc. The deformability of HT and control cells was evaluated using micropipette techniques, and the bulk viscosity of RBC suspensions at 40% hematocrit was measured. The shear elastic modulus, or "membrane rigidity", was more than doubled by heat treatment, although both the absolute value for mu and the estimate of the increase induced by heat treatment varied depending on the method of measurement. Heat treatment caused smaller increases in membrane viscosity and in membrane bending resistance, and only minimal changes in cell geometry. The deformability of HT cells was reduced: 1) the pressure required for cell entry (Pe) into 3 micrometers pipettes was increased, on average, by 170%; 2) at an aspiration pressure (Pa) exceeding Pe, longer times were required for cell entry into the same pipettes. However, when Pa was scaled relative to the mean entry pressure for a given sample (i.e, Pa/Pe), entry times were similar for control and HT cells. Bulk viscosity of HT RBC suspensions was elevated by approximately 12% on average (shear rates 75 to 1500 inverse seconds). These findings suggest that alteration of RBC membrane mechanical properties, similar to those induced by heat treatment, would most affect the in vivo circulation in regions where vessel dimensions are smaller than cellular diameters.     

Passive mechanical behavior of human neutrophils: power-law fluid.

The mechanical behavior of the neutrophil plays an important role in both the microcirculation and the immune system. Several laboratories in the past have developed mechanical models to describe different aspects of neutrophil deformability. In this study, the passive mechanical properties of normal human neutrophils have been further characterized. The cellular mechanical properties were assessed by single cell micropipette aspiration at fixed aspiration pressures. A numerical simulation was developed to interpret the experiments in terms of cell mechanical properties based on the Newtonian liquid drop model (Yeung and Evans, Biophys. J., 56: 139-149, 1989). The cytoplasmic viscosity was determined as a function of the ratio of the initial cell size to the pipette radius, the cortical tension, aspiration pressure, and the whole cell aspiration time. The cortical tension of passive neutrophils was measured to be about 2.7 x 10(-5) N/m. The apparent viscosity of neutrophil cytoplasm was found to depend on aspiration pressure, and ranged from approximately 500 Pa.s at an aspiration pressure of 98 Pa (1.0 cm H2O) to approximately 50 Pa.s at 882 Pa (9.0 cm H2O) when tested with a 4.0-micron pipette. These data provide the first documentation that the neutrophil cytoplasm exhibits non-Newtonian behavior. To further characterize the non-Newtonian behavior of human neutrophils, a mean shear rate gamma m was estimated based on the numerical simulation. The apparent cytoplasmic viscosity appears to decrease as the mean shear rate increases. The dependence of cytoplasmic viscosity on the mean shear rate can be approximated as a power-law relationship described by mu = mu c(gamma m/gamma c)-b, where mu is the cytoplasmic viscosity, gamma m is the mean shear rate, mu c is the characteristic viscosity at characteristic shear rate gamma c, and b is a material coefficient. When gamma c was set to 1 s-1, the material coefficients for passive neutrophils were determined to be mu c = 130 +/- 23 Pa.s and b = 0.52 +/- 0.09 for normal neutrophils. The power-law approximation has a remarkable ability to reconcile discrepancies among published values of the cytoplasmic viscosity measured using different techniques, even though these values differ by nearly two orders of magnitude. Thus, the power-law fluid model is a promising candidate for describing the passive mechanical behavior of human neutrophils in large deformation. It can also account for some discrepancies between cellular behavior in single-cell micromechanical experiments and predictions based on the assumption that the cytoplasm is a simple Newtonian fluid.     

Pituitary adenylate cyclase-activating peptide inhibits neutrophil chemotaxis

Pituitary adenylate cyclase-activating peptide 38 (PACAP 38) is a neuropeptide that displays several biological effects of interest in the context of airway diseases such as asthma and chronic obstructive pulmonary disease. These effects include inhibition of airway and vascular smooth muscle tone as well as modulation of inflammatory cell activity. However, little is known about the effect of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect neutrophil migration. A standard 48 well chemotaxis chamber was used to assess the effects of PACAP on N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced neutrophil chemotaxis and spontaneous random migration. PACAP 38 and VIP inhibited fMLP-induced human neutrophil chemotaxis. Furthermore, both peptides also exhibited a dose-related trend toward inhibiting the spontaneous, unstimulated migration of neutrophils. Since enhanced cell migration in cell chamber systems is reported to correlate with increased invasive properties in vivo, the presented inhibitory effects of PACAP 38 on neutrophil chemotaxis, supports the idea of an anti-inflammatory role for PACAP. This together with the well documented bronchodilatory capacity of PACAP might indicate a role for PACAP-agonists in future treatment of asthma and other inflammatory airway diseases.     

Rheology of passive and adhesion-activated neutrophils probed by atomic force microscopy

The rheology of neutrophils in their passive and activated states plays a key role in determining their function in response to inflammatory stimuli. Atomic force microscopy was used to study neutrophil rheology by measuring the complex shear modulus G*(omega) of passive nonadhered rat neutrophils on poly(HEMA) and neutrophils activated through adhesion to glass. G*(omega) was measured over three frequency decades (0.1-102.4 Hz) by indenting the cells 500 nm with a spherical tip and then applying a 50-nm amplitude multi-frequency signal. G*(omega) of both passive and adhered neutrophils increased as a power law with frequency, with a coupling between elastic (G') and loss (G') moduli. For passive neutrophils at 1.6 Hz, G' = 380 +/- 121 Pa, whereas G' was fourfold smaller and the power law coefficient was of x = 1.184. Adhered neutrophils were over twofold stiffer with a lower slope (x = 1.148). This behavior was adequately described by the power law structural damping model but not by liquid droplet and Kelvin models. The increase in stiffness with frequency may modulate neutrophil transit, arrest, and transmigration in vascular microcirculation.     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.     

Effect of lanthanides on red blood cell deformability and response to mechanical stress: role of lanthanide ionic radius

Prior studies exploring the effects of lanthanides (Ln) on red blood cells (RBC) have primarily focused on ion transport, cell fusion, and membrane protein structure. Our previous report [Biorheology 44 (2007), 361-373] dealt only with lanthanum (La) and cell rigidity; the present study extends these observations to other lanthanides (Nd, Sm, Eu, Dy, Er) and to RBC response to mechanical shear. Deformation-shear stress behavior of normal human RBC was measured at Ln concentrations up to 200 μM. In another series of experiments, RBC were exposed to mechanical stress (190 Pa, 300 s) at 50 μM Ln and deformation-stress data obtained prior to and after this stress. Data were fitted to a Lineweaver-Burke model to obtain the shear stress at one-half maximum deformation (SS1/2). Our results include: (1) lanthanides cause decreased cell deformability with the magnitude of the decrease dependent on concentration and shear stress; (2) this decrease of deformability is affected by Ln ionic radius such that La>Nd>Sm>Eu>Dy>Er and is reversible for cells in Ln-free media; (3) mechanical stress decreases deformability (i.e., increases SS1/2) such that compared to control, La and Sm reduce and Dy and Er enhance the mechanical stress effect; (4) the decrease of deformability consequent to mechanical stress scales inversely with Ln ionic radius. These results indicate a reciprocal relation between cell rigidity and sensitivity to mechanical stress that is mediated by Ln ionic radius. Additional studies are clearly warranted, particularly those that explore membrane-glycocalyx and intracellular mechanisms.     

Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry.

A magnetic bead microrheometer has been designed which allows the generation of forces up to 10(4) pN on 4.5 micron paramagnetic beads. It is applied to measure local viscoelastic properties of the surface of adhering fibroblasts. Creep response and relaxation curves evoked by tangential force pulses of 500-2500 pN (and approximately 1 s duration) on the magnetic beads fixed to the integrin receptors of the cell membrane are recorded by particle tracking. Linear three-phasic creep responses consisting of an elastic deflection, a stress relaxation, and a viscous flow are established. The viscoelastic response curves are analyzed in terms of a series arrangement of a dashpot and a Voigt body, which allows characterization of the viscoelastic behavior of the adhering cell surface in terms of three parameters: an effective elastic constant, a viscosity, and a relaxation time. The displacement field generated by the local tangential forces on the cell surface is visualized by observing the induced motion of assemblies of nonmagnetic colloidal probes fixed to the membrane. It is found that the displacement field decays rapidly with the distance from the magnetic bead. A cutoff radius of Rc approximately 7 micron of the screened elastic field is established. Partial penetration of the shear field into the cytoplasm is established by observing the induced deflection of intracellular compartments. The cell membrane was modeled as a thin elastic plate of shear modulus mu * coupled to a viscoelastic layer, which is fixed to a solid support on the opposite side; the former accounts for the membrane/actin cortex, and the latter for the contribution of the cytoskeleton to the deformation of the cell envelope. It is characterized by the coupling constant chi characterizing the elasticity of the cytoskeleton. The coupling constant chi and the surface shear modulus mu * are obtained from the measured displacements of the magnetic and nonmagnetic beads. By analyzing the experimental data in terms of this model a surface shear modulus of mu * approximately 2 . 10(-3) Pa m to 4 . 10(-3) Pa m is found. By assuming an approximate plate thickness of 0.1 micron one estimates an average bulk shear modulus of mu approximately (2 / 4) . 10(-4) Pa, which is in reasonable agreement with data obtained by atomic force microscopy. The viscosity of the dashpot is related to the apparent viscosity of the cytoplasm, which is obtained by assuming that the top membrane is coupled to the bottom (fixed) membrane by a viscous medium. By application of the theory of diffusion of membrane proteins in supported membranes we find a coefficient of friction of bc approximately 2 . 10(9) Pa s/m corresponding to a cytoplasmic viscosity of 2 . 10(3) Pa s.     

Shedding of the endothelial glycocalyx in arterioles, capillaries, and venules and its effect on capillary hemodynamics during inflammation

The endothelial glycocalyx has been identified as a barrier to transvascular exchange of fluid, macromolecules, and leukocyte-endothelium [endothelial cell (EC)] adhesion during the inflammatory process. Shedding of glycans and structural changes of the glycocalyx have been shown to occur in response to several agonists. To elucidate the effects of glycan shedding on microvascular hemodynamics and capillary resistance to flow, glycan shedding in microvessels in mesentery (rat) was induced by superfusion with 10(-7) M fMLP. Shedding was quantified by reductions of fluorescently labeled lectin (BS-1) bound to the EC and reductions in thickness of the barrier to infiltration of 70-kDa dextran on the EC surface. Red cell velocities (two-slit technique), pressure drops (dual servo-null method), and capillary hematocrit (direct cell counting) were measured in parallel experiments. The results indicate that fMLP caused shedding of glycans in all microvessels with reductions in thickness of the barrier to 70-kDa dextran of 110, 80, and 123 nm, in arterioles, capillaries, and venules, respectively. Intravascular volumetric flows fell proportionately in all three divisions in response to rapid obstruction of venules by white blood cell (WBC)-EC adhesion, and capillary resistance to flow rose 18% due to diminished deformability of activated WBCs. Capillary resistance fell significantly 26% over a 30-min period, as glycans were shed from the EC surface to increase effective capillary diameter, whereas capillary hematocrit and anatomic diameter remained invariant. This decrease in capillary resistance mitigates the increase in resistance due to diminished WBC deformability, and hence these concurrent rheological events may be of equal importance in affecting capillary flow during the inflammatory process.     

Aspiration of human neutrophils: effects of shear thinning and cortical dissipation.

It is generally accepted that the human neutrophil can be mechanically represented as a droplet of polymeric fluid enclosed by some sort of thin slippery viscoelastic cortex. Many questions remain however about the detailed rheology and chemistry of the interior fluid and the cortex. To address these quantitative issues, we have used a finite element method to simulate the dynamics of neutrophils during micropipet aspiration using various plausible assumptions. The results were then systematically compared with aspiration experiments conducted at eight different combinations of pipet size and pressure. Models in which the cytoplasm was represented by a simple Newtonian fluid (i.e., models without shear thinning) were grossly incapable of accounting for the effects of pressure on the general time scale of neutrophil aspiration. Likewise, models in which the cortex was purely elastic (i.e., models without surface viscosity) were unable to explain the effects of pipet size on the general aspiration rate. Such models also failed to explain the rapid acceleration of the aspiration rate during the final phase of aspiration nor could they account for the geometry of the neutrophil during various phases of aspiration. Thus, our results indicate that a minimal mechanical model of the neutrophil needs to incorporate both shear thinning and surface viscosity to remain valid over a reasonable range of conditions. At low shear rates, the surface dilatation viscosity of the neutrophil was found to be on the order of 100 poise-cm, whereas the viscosity of the interior cytoplasm was on the order of 1000 poise. Both the surface viscosity and the interior viscosity seem to decrease in a similar fashion when the shear rate exceeds approximately 0.05 s(-1). Unfortunately, even models with both surface viscosity and shear thinning studied are still not sufficient to fully explain all the features of neutrophil aspiration. In particular, the very high rate of aspiration during the initial moments after ramping of pressure remains mysterious.     

Transit time dispersion in pulmonary and systemic circulation: effects of cardiac output and solute diffusivity

We present an in vivo method for analyzing the distribution kinetics of physiological markers into their respective distribution volumes utilizing information provided by the relative dispersion of transit times. Arterial concentration-time curves of markers of the vascular space [indocyanine green (ICG)], extracellular fluid (inulin), and total body water (antipyrine) measured in awake dogs under control conditions and during phenylephrine or isoproterenol infusion were analyzed by a recirculatory model to estimate the relative dispersions of transit times across the systemic and pulmonary circulation. The transit time dispersion in the systemic circulation was used to calculate the whole body distribution clearance, and an interpretation is given in terms of a lumped organ model of blood-tissue exchange. As predicted by theory, this relative dispersion increased linearly with cardiac output, with a slope that was inversely related to solute diffusivity. The relative dispersion of the flow-limited indicator antipyrine exceeded that of ICG (as a measure of intravascular mixing) only slightly and was consistent with a diffusional equilibration time in the extravascular space of approximately 10 min, except during phenylephrine infusion, which led to an anomalously high relative dispersion. A change in cardiac output did not alter the heterogeneity of capillary transit times of ICG. The results support the view that the relative dispersions of transit times in the systemic and pulmonary circulation estimated from solute disposition data in vivo are useful measures of whole body distribution kinetics of indicators and endogenous substances. This is the first model that explains the effect of flow and capillary permeability on whole body distribution of solutes without assuming well-mixed compartments.     

Influence of 4-hydroxynonenal on chemiluminescence production by unstimulated and opsonized zymosan-stimulated human neutrophils

The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, fMet-Leu-Phe (fMLP) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast 10(-4) M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between fMLP and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different.     

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.     

Mechanical properties of peptidoglycan as determined from bacterial thread

Experiments are described in which the tensile strength, the extensibility and the initial Young's modulus of bacterial cell wall have been determined as functions of relative humidity in the range 11-98%. Data on stress relaxation and recovery are also given. Standard fibre-measuring technique has been used on 'bacterial thread', made from a cell-separation-suppressed mutant of Bacillus subtilis. The data show that peptidoglycan, the load bearing polymer in the cell wall, behaves very much like other viscoelastic polymers. Its mechanical behaviour when dry is that of a glassy polymer with tensile strength about 300 MPa and modulus about 20 GPa. When wet, it is weaker and much less stiff with tensile strength about 3 M Pa and modulus 10 M Pa. The relaxation data indicate a wide spectrum of relaxation times. The results are discussed in terms of the structure of peptidoglycan and its orientation in the bacterial cell wall. The way in which mechanical behaviour depends strongly on humidity is compared with that of other biopolymers in terms of possible hydrogen-bond density and the ordering of water molecules. The possibility of a well-defined glass transition is briefly examined.