rheological and mechanical aspects of leukocytes …...a. alexandrova et al./ rheological and...

Series on Biomechanics, Vol.27, No. 3-4 (2012), 74-79

74

Rheological and mechanical aspects of leukocytes motion and adhesion

A. Alexandrova, N. Antonova

Department of Biomechanics, Institute of Mechanics, Bulgarian Academy of Sciences, Acad. G.

Bonchev str., Bl.4, 1113 Sofia, Bulgaria; [email protected]; [email protected]

Abstract

The questions of leukocyte adhesion to endothelium and motion of leukocytes in blood flow are important to treatment of many diseases occurring at vascular level. In the present study an overview of the literature of cell mechanics and cell adhesion as a multistage process that influence the microcirculation is done. These processes are characterized with some parameters, as a mean blood flow velocity, a critical velocity, a wall shear rate, a wall shear stress, etc. It is found in many of the studies that the investigated parameters are close related with the changes under pathological conditions. The investigation of the interaction of leukocyte adhesion molecules and the endothelial receptors may help our understanding of leukocyte behavior in the main vascular dysfunctions. There are different technical approaches used in current practice to study leukocyte adhesion under flow in real time – in vitro flow chamber (FCA) and in vivo intravital microscopy (IVM).

Keywords: Leukocyte rheology, leukocyte-endothelium interactions, flow chamber assays (FCA), intravital microscopy (IVM) _________________________________________________________________________________________

1. Introduction In inflammatory processes in response to various activations induced by the protecting mechanisms

against foreign organisms, different kinds of leukocytes undergo cytoskeleton and/or membrane changes. These changes involve among others the modification of rheological properties of leukocytes such as the deformability, the adhesiveness to the endothelium and the generation of inner force for transmigration (diapedesis) [1]. However, changes in the mechanical and adhesive properties control the circulation of leukocytes, and may contribute to the pathology of cardiovascular disorders, atherosclerosis, diabetes, est. Attention has focused on the concept to define circulatory factors that cause disease and factors which affect clinical outcome. Such studies are dependent on development of procedures for measurement of rheological parameters [2]. 2. Leukocyte rheology 2.1. Cellular mechanics

Blood flow in the microcirculation depends on the pressure generated by the contraction of the heart muscle. The capillaries in many organs are narrower than the dimensions of red cell and the leukocytes. The cells have to deform in order to fit into the lumen; therefore their mechanical properties have an important influence on microcirculation. Thus the resistance to flow in capillaries depends on the deformability of the blood cells and the endothelium. The deformability is dependent on the properties of their components [3].

The WBC membrane has numerous folds – microvilli, which provide an excess area about 100%, which probably allows cell deformation without a change in surface area and volume [1]. Whereas erythrocytes consist of a viscoelastic membrane with cytoplasm of relatively low and constant viscosity, all

A. Alexandrova et al./ Rheological and mechanical aspects of leukocytes motion and adhesion

75

leukocytes exhibit viscoelastic properties depend on internal structures. Leukocytes stiffness rigidity is largely determined by the cytoskeleton of actins and actins-binding protein and its degree of cross linking [2]. Together with the fact that white blood cells have a larger volume than red cells this leads to a situation that the resistance imposed by a single white cell in capillaries is much larger than that of a single red cell. Fortunately, we have much fewer leukocytes than erythrocytes in the circulation [3]. The viscous coefficient is several orders of magnitude higher than that of erythrocytes, and this is manifest in ability of leukocytes to cause intermittent cessation of capillary flow [2].

The rheological changes allow enable the defence against foreign organisms to be controlled, but also contribute to the microcirculatory failure. During local or systematic ischemia, microcirculatory obstruction is facilitated by the reduced WBC deformability and increased adhesion [1]. Stimulation with agents such as bacterial peptides, activated complements, cigarette smoke and auto antibodies make leukocytes markedly more resistant to deformation [2]. The circulating leukocytes become trapped in the microcirculation during hemorrhagic shock and play a key role in the initiation of oxygen free radical formation, cell injury, thrombogenesis and organ failure [1].

2.2. Cell adhesion

In the circulation leukocytes participate in the inflammatory and immune responses, and are the first cells adhering to microvascular endothelium. The process can be considered in some aspects as a set of rheological phenomena:

- Migration of leukocytes in blood flow and hence collision with vessel wall depends on the direct hemodynamic interaction of leukocytes with erythrocytes in postcapillary venules – erythrocytes push leukocytes against the endothelium.

- Attachment to endothelium occurs in the presence of flow, so that initial capture depends on the relative kinetics of bond formation and cell motion, as well as leukocyte deformability, while attachment of bond depends on the shear stress imposed on the adherent cell [2].

Cellular adhesion is made possible by the so-called “adhesion molecules” which enable interactions between endothelium cells and white blood cells. Three major groups of molecules mediate adhesion:

- Мembers of the selectin; - Heterodimeric integrins; - Immunoglobulin super-family. Leukocyte adhesion to the inflamed vessel wall of postcapillary venules begins with a capture of free

WBCs to the endothelium. Leukocyte capture is followed by leukocyte rolling along the endothelium. Both biological processes - capture and rolling, are mediated by selectins (including the three selectins P-, E-, and L-selectin), and other adhesion molecules, binding to their counter-receptors (see Fig. 1). While rolling, leukocytes come into close contact with the inflamed endothelium, enabling them to interact with endothelium-bound chemokines. Chemokines bind to specific chemokine receptors on the leukocyte surface, which triggers the activation of integrins, leading to a film of adherent leukocytes arrested on the endothelium (the most important adhesion molecules of immunoglobulin super-family are ICAM-1, ICAM-2, VCAM-1, which act are ligands for integrins LFA-1 and VLA-4) and eventual transmigration through the endothelium into tissue [4].

The quantity of leukocyte adhesion molecules and of their receptors assists understanding of leukocyte behavior in vascular diseases, along with measurement of flow resistance of leukocytes. Vascular cell adhesion molecule (VCAM-1) is abnormally present on endothelial cells in atherosclerosis, diabetes mellitus and inflammatory conditions. Soluble forms of intracellular adhesion molecules (ICAM-1) or VCAM can be found elevated in blood of patients with rheumatoid arthritis or infections disease [2]. 3. Characteristics of motion of WBCs at adhesion process

To understand the mechanism of leukocyte adhesion, it is important to recall the physical conditions

under which WBCs can adhere in blood flow conditions.


76

Fig.1. Leukocyte - endothelium interaction [10] - Blood flow velocities profiles in blood vessels are slightly blunted parabolas, with higher blood flow

velocities near to the centre of the vessel (“centreline velocity”, VCL, or “maximal” velocity, Vmax), while flow velocity decreases towards the vessel wall.

- The mean blood flow velocity (V blood ) is calculated according to the formula:

(3.1.) 2.2max

DD

VV

VLblood

(µm.s-1),

where V max is the velocity of the fastest noninteracting leukocyte, DL the leukocyte diameter, and DV the vessel diameter (see Fig. 2A).

- The lowest velocity of noninteracting leukocytes at the margin of a given vessel is called a critical velocity (V crit ). If the cell at the vessel margin moves at a velocity:

a) Below V crit , it is slower down by adhesive interaction with endothelial ligands; b) Above or equal to V crit , it is considered to be non interacting. - When the marginated leukocytes move at a velocity below V crit and bind with endothelium, the

continuous blood flow exerts considerable shear forces on interacting cells. This is due to the difference in velocity between the slower moving or adherent leukocytes and faster moving blood (see Fig. 2B).

- To find the force acting on rolling or adherent leukocytes, the wall shear rate (WSR) . can be calculated

for each venule by equation: (3.2.) DV Vblood 8. (s-1), where Vblood is the mean blood flow velocity and Dv is the vessel diameter.

(A) (B) (C)

Fig.2. (A) Velocity profile in blood vessel. (B) Pro- and antiadhesive forces in blood. (C) Electrostatic repulsion [6]

- The wall shear stress (WSS) can be calculated by equation:

(3.3.) .

. (mPa.s),


77

where is the fluid viscosity (viscosity depend on the hematocrit and the vessel diameter due to erythrocyte

deformability), and . is the wall shear rate (WSR). When cells enter postcapillary venules in inflamed

tissue, they need to rapidly make contact with (or “tether”) and arrest on endothelium before being carried away by continuous blood flow.

- When leukocytes pass through capillaries of similar or smaller diameter than their own, they often move more slowly than red blood cells. When entering larger postcapillary vessels, red blood cells lined up behind slower moving leukocytes, flow past the leukocytes, thus pushing them towards the endothelium – force margination (see Fig.2B).

- On the other hand, leukocytes are the biggest objects in blood and therefore tend to flow near to the centre of a blood vessel (a process known as “dispersion”), where velocity flows is highest (see Fig. 2B).

- Negatively charged membranes of leukocytes and endothelium also reduce adhesion by electrostatic repulsion (see Fig. 2C).

The wall shear stress describes the viscous drag of liquids on adherent leukocytes situated on vessel walls. It increases with rising flow velocity and liquid viscosity, and with decreasing vessel diameter. To overcame physical constraints imposed by hemodynamic parameters, i.e., high shear stresses, dispersion of the leukocytes, and electrostatic repulsion, while the same at time allowing subset-specific recruitment, leukocyte adhesion has evolved as a multistep process [6].

Table 1

Techniques to study leukocyte adhesion: FCA and IVM.

Flow chamber assays (FCA) Intravital microscopy (IVM)

Equ

ipm

ent

and

a w

ay o

f wor

k

Flow chambers are assembled from disposable floor, such as Petri dish, coated with purified adhesion molecules. The flow chamber is mounted on top of adhesive spot and held in place by application of vacuum (see Fig. 3). Alternatively, capillary flow chambers can be used. Assembled flow chambers are placed on an inverted microscope connected to a video system. Using a precision syringe pump, cells are passed through the flow chamber under defined shears, and events are recorded for off-line analysis.

Fig.3. Experimental setups for studying leukocyte adhesion under physiological shear flow. (A) Transwell-based assay used for the assessment of leukocyte transendothelian migration. (B) Standard flow chamber setup [5].

To a specialized microscope are transferred animals after surgical preparation, which is placed upright (in some cases inverted) and vascular bed is visualized by bright-field transillumination, or by bright-field/fluorescent epi-illumination (seeFig.4). In the latter configuration, light comes through the object lens and reflected light is collected via the same lens. Events are recorded using a high-sensitivity video-speed camera, processed using an image processor, and record for off-line analysis of interaction parameters.

Fig.4. (A) IVM setup. (B) The low- and high-magnification image of the inguinal LN microvasculation after injection of the plasma marker [6].


78

App

licat

ion

-To recreate all aspects of leukocyte recruitment, from rolling interaction to film adhesion and transmigration; -to measure parameters: DV, Vblood, γ, τ, total cellular flux, rolling flux, fast cell velocity, rolling velocity, shear resistance (number of adherent cells after each increase in shear rate), and other; -biophysical measurements of adhesion receptor binding kinetics, such as L-selectin on and off rates.

-Endogenous or exogenous cells can be observed in IVM; -transillumination (i.e., light coming from a bright-field source from below the specimen) is only suitable in thin tissues; used to visualize the general anatomy of vascular beds and the study of cell-endothelium interaction; -bright-field/fluorescent illuminescent microscopy is used to study cells in thick tissues.

Adv

anta

ges

-Precise control of the adherent molecules and shear force; -the easy of manipulation of observed cells; -can be used to reconstruct artificial endothelial surface using specific protein preparations; -by combining various adhesion receptor and adhesion-triggering molecules such as chemokines, the exact contribution of each element can be critically examined; -a large number of adhesive interactions can be tracked simultaneously in FCAs, as the interactive area generally covers the entire field of view, and all events take place in the same focal plane; -FCA does not require microsurgical manipulation of animals; -FCAs used to dissect molecular events during integrin activation and transmigration, using cells expressing fluorescent proteins or other probes that can be followed by sensitive video camera system; -flow chambers also present a number of differences from blood vessels.

-IVM is the directed, real-time in vivo observation of leukocyte - endothelium interaction in a wide variety of vascular beds; -IVM assays not only re-examine in vitro data under physiological conditions, but allow the discovery an often unexpected and remarkable specialization of postcapillarly venules in leukocyte recruitment.

Lim

itatio

ns

-Contact between the bottom chamber and flowing leukocytes is only mediated by gravity, the shear forces than can be applied in flow chamber are necessarily limited to 1,5-2 dyn.cm-2, except when shear resistance is being tested (where the flow is deliberately stopped before gradually an creasing to > 10 dyn.cm-2) or when transmigration of adherent cells is studied; -it is difficult to assess whether the adhesion receptor densities used in FCAs correspond to the densities encountered under physiological circumstances; -when studying transmigration, has to consider the large variety of endothelial phenotypes, and transmigration mechanisms are differ between postcapillary endothelial cells in different microvascular beds [5, 6, 7, 8, 9].

-Limitation of epifluorescent IVM is quenching of excitation and emission light by tissue factors, especially hemoglobin; this is translates into two major disadvantages: first, the penetration of depth for IMV is often low, such that deeper blood vessels cannot be really visualized, and second, weak florescent signals, such as emitted by fluorescent labels proteins are often difficult to detect, which limits analysis of molecular dynamics; -interacting leukocyte subset (e.g., neutrophils or monocytes) cannot usually be identified with transilluminescent IVM, although after experiment used immunohistogy; fast-moving cells cannot be individually discerned and thus the fraction of interacting cells out of total cells cannot be determined [5, 6, 7, 8, 9].

4. Techniques to study leukocyte adhesion under flow and in real time

The adhesion of leukocyte is a dynamic process, which happens under flow and time. For this reason

the experimental methods (micropipette technique, viscoelastic testing technique, atomic force microscopy, filtration analysis) for investigating of leukocyte rheology, which measure the deformability and recovery of leukocytes at their movement in the microcirculation are not adequate for quantitating the adhesion of WBCs [5, 6, 7, 8, 9]. For analysis of the adhesion of flowing leukocytes under (near-) physiological conditions are used flow chamber assays (FCA) and intravital microscopy (IVM) (see Table 1).


79

5. Conclusion

The leukocytes play important biological functions connected with blood rheology. Rheological properties of the leukocytes influence their migration in the blood stream and WBCs contacts with the endothelial cells in the micro-vessel walls. The formation of adhesive bonds depends on local velocity and shear forces to the captured cells. The methods of FCA and IVM could characterize and quantify processes as WBC adhesion/rolling, chemokine signalling, integrin activation and leukocyte transmigration through micro-vessel walls in health and diseases. References [1] Stoltz, J.-F., Singh, M., Riha, P., Rheology of Leukocytes, 36-48.In the book: Stoltz, J.-F., Singh, M., Riha, P., 1999. Hemorheology in Practice. IOS Press, Netherlands [2] Wautier, J.-L., Schmid-Schonbein, G. W., Nash, G. B., 1999. Measurement of leukocyte rheology in vascular disease: clinical rationale and methodology. Clinical Hemorheology and Microcirculation 21, 7-24. [3] Geert, W. S.-S., Cell activation in the circulation: The auto-digestion hypothesis, 131-145.In: Chien, Shu, Chen, P. C. Y., Fung, Y. C. (Eds.), 2008. An Introductory Text to Bioengineering. Word Scientific, Singapore. [4] Sperandio, M., Walzog, B., Mechanisms of inflammation: Neutrophils, 248-268. In: Hamann, Alf, Engelhardt, Br. (Eds.), 2005. Leukocyte Trafficking. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. [5] Alon, R., Cinamomn, G., Luscinskas, F. W., Real Time in Vitro for Studying Leukocyte Transendotheial Migration under Physiological Flow Conditions, 437-454. In: Hamann, Alf, Engelhardt, Br. (Eds.), 2005. Leukocyte Trafficking. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. [6] Stein, J.V., 2005. Intravital Microscopy and In Vitro Flow Chamber: Techniques to Study Leukocyte Adhesion under Flow and in Real Time, 455-471. In: Hamann, Alf, Engelhardt, Br. (Eds.) Leukocyte Trafficking. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. [7] Tran-Son-Tay, R., Nash, G.B., 2007. Mechanical Properties of Leukocytes and Their Effects on the Circulation, 137-152. In: Baskurt, O. K., Hardeman, M. R., Rampling, M. W., Meiselman, H.J. (Eds.), Handbook of Hemorheology and Hemodynamics. IOS Press, Netherlands. [8] Cranmer, S.L., Nash, G.B., Adhesion of Circulating Leukocytes and Platelets to the Vessel Wall, 153-169.In: Baskurt, O. K., Hardeman, M. R., Rampling, M. W., Meiselman, H.J. (Eds.), 2007. Handbook of Hemorheology and Hemodynamics. IOS Press, Netherlands. [9] Sundd, Pr., Pospieszalska, M. K., Cheung, L. S.-L., 2011. Biomechanics of Leukocyte Rolling. Biorheology 48, 1-35. [10] Gough, M. V., Kyriakides, C., Hechtman, B.H., 2002. ACS Surgery: Principles and Practice, Section 8 Chapter 26: Molecular and cellular mediators of the inflammatory response. Web MD Inc.

rheological and mechanical aspects of leukocytes …...a. alexandrova et al./ rheological and...

Documents