influence of local haemodynamics on leucocyte rolling and chemoattractant-induced firm adhesion in...

In¯uence of local haemodynamics on leucocyte rolling

and chemoattractant-induced ®rm adhesion

in microvessels of the rat mesentery

X . X I E , P . H E D Q V I S T and L . L I N D B O M

Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden

ABSTRACT

Tissue hyperaemia, oedema formation and leucocyte accumulation are characteristic features of the

inflammatory process referable to changes at the microcirculatory level. Here, we used intravital

fluorescence video microscopy to assess relationships among haemodynamical parameters,

leucocyte rolling, and chemoattractant-induced firm adhesion in small venules (13)24 lM) of the rat

mesentery. The rolling leucocyte ¯ux in these vessels was directly proportional to the total leucocyte

¯ux (r � 0.76, P < 0.001), which in turn closely correlated to the venular blood ¯ow (r � 0.77,

P < 0.001). Consequently, the rolling to total leucocyte ¯ux fraction, averaging 39 � 15%, did not

vary with the blood ¯ow and showed no correlation to either blood ¯ow velocity (r � )0.15,

P � 0.42) or wall shear rate (r � )0.06, P � 0.77), indicating that the extent of leucocyte rolling is

not primarily dependent on the ¯uid viscous drag at physiological blood ¯ow rates in vivo. Stimulation

of the mesentery with the chemoattractant fMLP (10)6M) induced ®rm adhesion of rolling

leucocytes. It was found that the number of adherent leucocytes in individual vessels was directly

related to the rolling leucocyte ¯ux (r � 0.78, P < 0.001) and hence to the venular blood ¯ow

(r � 0.47, P < 0.05), while there was no correlation to the wall shear rate (r � 0.27, P � 0.24). The

dependence of the ®rm adhesive response on the blood ¯ow level and the delivery rate of leucocytes

was con®rmed at the whole organ level. Thus, leucocyte accumulation in rat skin lesions was

markedly enhanced when a vasodilator was co-administered with the chemotactic stimulus

compared with chemotactic stimulation alone. The data indicate that, within a physiological blood

¯ow range, the leucocyte response to chemotactic stimulation is largely independent of the prevailing

hydrodynamic shear forces. Instead, manifestation of the ®rm adhesive response, because of its

dependence on the preceding rolling interaction, is clearly related to the blood ¯ow level in the

microvessels, which emphasizes the signi®cance of tissue hyperaemia in in¯ammation.

Keywords blood ¯ow, in¯ammation, intravital microscopy, leucocyte/endothelium interactions, rat

mesentery.

Received 3 February 1998, accepted 5 October 1998

Redness, heat and tissue swelling are clinical signs of

in¯ammation which can be referred to changes at the

microcirculatory level, namely arteriolar dilatation

leading to increased local blood ¯ow, plasma exudation

resulting from increased vascular permeability, and

accumulation of circulating leucocytes. A complex

pattern of mediator actions governs these microvas-

cular events, which in turn mutually may in¯uence

each other. Thus, hyperaemia may potentiate not only

¯uid transfer across the vessel wall but also the

recruitment of leucocytes to the extravascular tissue. It

is clearly indicated by data obtained in various in vivo

models that co-stimulation with vasodilating agents

and chemotactic mediators results in enhanced leuco-

cyte accumulation compared with chemotactic stimu-

lation alone (Issekutz 1981, Issekutz & Movat 1982,

Downey et al. 1988, Raud et al. 1988, Texieira et al.

1993), presumably due to increased delivery rate of

leucocytes with increasing tissue blood ¯ow. On the

other hand, high microvascular ¯ow rates may in¯u-

ence the leucocyte extravasation process unfavourably,

as an increased hydrodynamic force will make it more

dif®cult for the leucocyte to establish ®rm contact with

the vessel wall.

Correspondence: Lennart Lindbom, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden.

Acta Physiol Scand 1999, 165, 251±258

Ó 1999 Scandinavian Physiological Society 251

Leucocyte extravasation is a multistep process

accomplished through sequential adhesion receptor-

dependent interactions with vascular endothelium and

extravascular matrix components (Butcher 1991, Carlos

& Harlan 1994). Slow rolling of the leucocytes along

the venular endothelium, mediated by the selectin

family of adhesion molecules, is the initial event in this

process and a necessary precondition for subsequent

(integrin-mediated) ®rm attachment and transmigration

(Lawrence & Springer 1991, Lindbom et al. 1992, von

Andrian et al. 1992). Through the rolling interaction,

retardation of the fast-¯owing leucocyte is achieved,

enabling the leucocyte to establish ®rm contact with the

vessel wall in the presence of high ¯uid shear. Although

binding via the selectins appears to be relatively shear-

resistant, inverse relationships between leucocyte roll-

ing and wall shear rate has been indicated both in vitro

(Lawrence et al. 1987, Lawrence & Springer 1991) and

in vivo (Atherton & Born 1973, Firrell & Lipowsky 1989,

Ley & Gaehtgens 1991, Damiano et al. 1996). In view

of the close relationship between the extent of leuco-

cyte rolling and the ®rm adhesive response induced by

chemoattractants (Lindbom et al. 1992), these data

suggest that leucocyte adhesion, and hence recruitment

to tissues, will be impeded by increased ¯uid shear.

However, such an implication is dif®cult to reconcile

with the ®ndings of the functional studies referred to

above, i.e. potentiation of leucocyte recruitment to

tissues by increased blood ¯ow.

The present study aimed at investigating relation-

ships between local haemodynamics and leucocyte/

endothelium interactions (leucocyte rolling and ®rm

adhesion) in small venules of the rat mesentery. Direct

intravital microscopic observations in this model

showed that the leucocyte rolling fraction did not vary

in parallel with the blood ¯ow or the wall shear rate in

the microvessels. Instead, there was a proportional

relationship between rolling leucocyte ¯ux and venular

blood ¯ow. In congruity, because of its dependence on

the preceding rolling interaction, the ®rm adhesive

response to chemoattractant stimulation was found to

also correlate with the microvessel blood ¯ow. These

data emphasize the signi®cance of tissue hyperaemia in

in¯ammation for manifestation of the leucocyte

response in host defense and immune reactions.

MATERIALS AND METHODS

Animals

Adult Wistar rats were used in this study. The rats were

anaesthetized with equal parts of ¯uanison/fentanyl

(Hypnorm, 10/0.2 mg mL)1; Janssen Pharmaceutica,

Beers, Belgium) and midazolam (Dormicum,

5 mg mL)1; Hoffman-La Roche, Basel, Switzerland)

diluted 1:1 with sterile water [0.2 mL (100 g body

wt))1 intramuscularly]. The trachea was cannulated to

facilitate spontaneous breathing. A catheter was placed

in the left femoral vein or the left jugular vein for i.v.

administration of supplementary doses of anaesthesia.

Body temperature was maintained at 37 °C by a heating

pad connected to a rectal thermistor.

Intravital microscopy

Laparotomy was performed by a midline incision, and a

segment of the ileum was exteriorized from the peri-

toneal cavity and placed on a heated transparent ped-

estal to allow microscopic observation of the

mesenteric microcirculation. The exposed tissue was

superfused with a thermostated (37 °C) bicarbonate-

buffered saline solution (composition in mM: NaCl 132,

KCl 4.7, CaCl 2.0, MgSO4 1.2, NaHCO3 18) equili-

brated with 5% CO2 in nitrogen to maintain physio-

logical pH. Observations of the mesenteric

microcirculation were made using a Leitz Orthoplan

microscope equipped with water immersion lenses

(´ 25, NA 0.6; ´ 55, NA 0.8). The microscopic image

was televised (Panasonic WV-1550, Panasonic WV-

1900 low light camera) and recorded on video tape

(Panasonic NV-F 100 S-VHS) via a time/date genera-

tor (Panasonic WJ-810) for subsequent off-line analysis.

After positioning under the microscope, a 20±30 min

stabilization period preceded quantitative measure-

ments. In order to detect the free-¯owing leucocytes in

the observed vessel segments, acridine orange

[5 mg (kg body wt))1; Sigma Chemical, St. Louis, MO]

was given intravenously to label the circulating leuco-

cytes, and short periods of ¯uorescent light epi-illumi-

nation (Leitz Ploemopak, ®lter block I2) was applied.

Analyses of blood ¯ow parameters, leucocyte ¯ux and

leucocyte/endothelium interactions (rolling and ®rm

adhesion) were made in small venules (inner diameter

15±25 lm) with stable resting blood ¯ow.

Determination of leucocyte ¯ux and blood ¯ow parameters

Rolling leucocyte ¯ux in the venules was determined

from the recorded video images by counting the num-

ber of rolling leucocytes per minute passing a reference

point in the microvessel under normal light transillu-

mination. The corresponding ¯ux of free-¯owing leu-

cocytes was similarly determined in the ¯uorescent

scenes. The leucocyte rolling fraction was calculated as

the rolling cell ¯ux in percentage of the total leucocyte

¯ux (rolling plus free-¯owing cells). Individual leucocyte

rolling velocities were calculated from the time required

for steady rolling leucocytes to travel a de®ned distance

(100±200 lm) in the microvessel. Distance was mea-

sured by hand with a caliper directly on the TV-screen.

Microvascular blood ¯ow and leucocyte/endothelium interactions � X Xie et al. Acta Physiol Scand 1999, 165, 251±258

252 Ó 1999 Scandinavian Physiological Society

The mean rolling velocity in each vessel was determined

as the average of »10 leucocytes passing in the venule.

Detection of the free-¯owing leucocytes in ¯uorescent

light also permitted, through frame-by-frame analysis,

determination of the velocity of individual free-¯owing

leucocytes by measuring the distance travelled between

two or more successive video frames. The highest cell

velocity among >15 leucocytes analysed in a given

moment was considered to correspond to the maximal

¯ow velocity (vmax) in the vessel, and was used to esti-

mate the mean blood ¯ow velocity (vmean) according to

the relationship vmean � vmax/{2 ) (//2r)2} where /represents leucocyte diameter (»7 lm for the rat) and r

is the vessel radius (Ley & Gaehtgens 1991). Mean

blood ¯ow velocity and vessel radius were used to cal-

culate volume blood ¯ow (Q , nL s)1) and wall shear

rate (c, s)1) according to the equations: Q � pr2vmean

and c � 8vmean/2r, respectively. Wall shear stress

(dyn cm)2) was calculated as the product of viscosity

and shear rate, assuming a constant viscosity of 0.025 P

(Lipowsky et al. 1980).

Leucocyte ®rm adhesion

After recording for analysis of blood ¯ow and leucocyte

¯ux in resting tissue, leucocyte ®rm adhesion was

induced by the chemotactic peptide N-formyl-

methionyl-leucyl-phenylalanine (fMLP; Sigma) admini-

stered topically to the mesentery for 6 min via the

superfusion buffer at a ®nal concentration of 10)6M.

At the end of the stimulation period, the number of

adherent leucocytes (stationary for >1 min) per

100 lm venule length was counted. During the fMLP

stimulation period, rolling velocity of the leucocytes

which became ®rmly attached within the ®eld of

observation was determined through reverse video

play-back analysis of the distance travelled by these cells

every half second prior to their ®rm arrest. Rolling

velocity of those leucocytes which did not adhere

within the same venular segments was similarly deter-

mined.

Skin lesion experiments

In a different group of experiments, skin lesions were

induced in four rats with their backs shaved 16 h prior

to the experiment. In these experiments, zymosan-

activated rat plasma (ZAP) was used as the chemotactic

stimulus. Plasma of heparinized rat blood was incu-

bated with zymosan (10 mg mL)1; Sigma) for 60 min

at 37 °C resulting in complement activation and gen-

eration of C5a. Intradermal injections (100 lL) of PBS,

prostaglandin E2 (PGE2, 10)6M), ZAP (undiluted), or

ZAP plus PGE2 were made in duplicate at alternate

sites in the dorsal region of the animals. The animals

were killed 4 h later with an overdose of anaesthetic,

and the skin lesions were carefully removed and dis-

sected free from fat and muscle tissue. For quanti®ca-

tion of leucocyte in®ltration in the skin lesions, the skin

samples were homogenized in 10 mL 0.5% hex-

adecyltrimethylammonium bromide, freeze-thawed and

centrifuged, whereafter the myeloperoxidase (MPO)

activity of the supernatant was determined. MPO is

abundant in neutrophils and found to be a reliable

marker for neutrophil accumulation in in¯amed tissues

(Lundberg & Arfors 1983). The enzyme activity was

determined spectrophotometrically as the change in

absorbency at 650 nm (20 °C) that occurs in the redox

reaction of H2O2-tetramethylbenzidine catalysed by

MPO, and expressed as units MPO g tissue)1.

Statistics

Values are presented as mean � SD. Calculation of

statistical signi®cance was performed with the

Wilcoxon test for paired samples and the Mann±

Whitney U-test for independent samples. Correlation

between variables was assessed according to Pearson's

product moment correlation.

RESULTS

Leucocyte/endothelium interactions and blood ¯ow

parameters were measured in small venules of the rat

mesentery exposed for intravital microscopy. A total of

30 venules (mean i.d., 18.2 � 3.5 lm; range,

13±24 lm) in 15 rats were used for analysis. Blood ¯ow

velocity in these vessels ranged from 0.1 to 1.4 mm s)1

(mean, 0.7 � 0.3 mm s)1) yielding wall shear rates and

blood ¯ows in the range 46±558 s)1 (mean,

288 � 116 s)1) and 0.02±0.45 nL s)1 (mean, 0.19 �

0.14 nL s)1), respectively. Correspondingly, calculated

wall shear stress varied between 1.2 and 14.0 dyn cm)2

(mean, 7.2 � 2.9 dyn cm)2 (Table 1).

Correlation analysis of ¯ow parameters in individual

vessels revealed that the total leucocyte ¯ux (rolling

plus free-¯owing cells) in the venule was closely related

to the venular blood ¯ow (r � 0.77, P < 0.001). The

rolling leucocyte ¯ux in these vessels ranged from 2 to

53 cells min)1 and was directly proportional to the total

leucocyte ¯ux (r � 0.76, P < 0.001) (Fig. 1a), and,

accordingly, related also to the blood ¯ow (r � 0.51,

P < 0.01) (Fig. 1b). Thus, within the physiological

blood ¯ow range observed, the rolling to total leucocyte

¯ux fraction, averaging 39 � 15% (range, 18±75%), did

not vary in parallel with the venular blood ¯ow

(r � )0.22, P � 0.25). Moreover, leucocyte rolling

fraction showed no correlation to either blood ¯ow

velocity (r � )0.15, P � 0.42) or wall shear rate

(r � )0.06, P � 0.77) (Fig. 2a), indicating that the


Acta Physiol Scand 1999, 165, 251±258 X Xie et al. � Microvascular blood ¯ow and leucocyte/endothelium interactions

extent of leucocyte rolling is not primarily dependent

on the hydrodynamic force exerted by the blood stream

at physiological ¯ow rates in vivo. Likewise, mean leu-

cocyte rolling velocity, ranging 5.1±37.6 lm s)1

(19.7 � 7.7 lm s)1), showed no correlation to blood

¯ow velocity (r � 0.14, P � 0.48) or wall shear rate

(r � 0.22, P � 0.25) (Fig. 2b).

In resting tissue, only occasional cells were found to

spontaneously adhere to the venular endothelium

during observation periods of 1±2 h. Stimulation with

fMLP (10)6M) induced prompt leucocyte adhesion,

resulting in 16.5 � 7.8 adherent cells per 100 lm ve-

nule length. The number of adherent cells in individual

vessels correlated strongly with the rolling leucocyte

¯ux (r � 0.78, P < 0.001) (Fig. 3) and to a lesser ex-

tent with the venular blood ¯ow (r � 0.47, P < 0.05).

On the other hand, there was no correlation to the wall

shear rate (r � 0.27, P � 0.24). It was found that

leucocytes which adhered to the endothelial lining

(®rmly attached for >1 min) were recruited exclusively

from the rolling population. Moreover, there was a

clear predominance of slowly rolling cells among those

leucocytes which were found to adhere. Thus, the

rolling velocity of those leucocytes which came to arrest

in the observed vessel segment was on average

12.8 � 6.8 lm s)1 whereas leucocytes which did not

adhere, but continued to roll in this segment in its

whole length, were rolling with a signi®cantly higher

velocity, i.e. 32.1 � 16.4 lm s)1 (P < 0.001) (Fig. 4).

Typically, the ®rm adhesive event was not preceded by

Figure 1 Rolling leukocyte ¯ux vs. total leucocyte ¯ux (a) and blood

¯ow (b) in rat mesenteric venules. Linear regression line is superim-

posed.

Figure 2 Leucocyte rolling fraction (a) and leucocyte rolling velocity

(b) as a function of wall shear rate in rat mesenteric venules. Each

data point in panel (b) represents the average velocity of »10 rolling

leucocytes analysed in each venule. Linear regression line is super-

imposed.

Table 1 Microvascular and leucocyte parameters in rat mesenteric

venules

Blood ¯ow velocity (mm s±1) 0.66 � 0.31 (0.09±1.39)

Diameter (lm) 18.2 � 3.5 (13±24)

Wall shear rate (s±1) 288 � 116 (46±558)

Wall shear stress (dyn cm±2) 7.2 � 2.9 (1.2±14.0)

Volume blood ¯ow (nL s±1) 0.19 � 0.14 (0.02±0.45)

Total leucocyte ¯ux (cells min±1) 54 � 29 (10±118)

Rolling leucocyte ¯ux (cells min±1) 20 � 13 (2±53)

Leucocyte rolling fraction (%) 39 � 15 (18±75)

Leucocyte rolling velocity (lm s±1) 19.7 � 7.7 (5.1±37.6)

Values are mean � SD. Range is shown in brackets.



a gradual decline in rolling velocity, but rather, rolling

velocity was quite uniform until 1±2 s prior to the ®rm

arrest. The relationship between rolling velocity and

®rm attachment suggests that a critical rolling velocity

level exists which must not be exceeded in order to

permit transition from a rolling to a ®rm adhesive state.

By statistical means, under the present conditions in the

rat mesentery, an upper limit for rolling velocity at

26 lm s)1 (given by the mean + 2SD) would encom-

pass >95% of the cells capable of adhering in the small

venules studied.

The observed relationship between the (chemoat-

tractant-induced) ®rm adhesive response and the

delivery rate of leucocytes in individual microvessels of

the rat mesentery clearly suggests, if extrapolated to the

whole organ level, that tissue blood ¯ow is an

important factor in the regulation of leucocyte

recruitment to in¯amed tissue sites. In separate

experiments, the existence of such relationship in rat

tissue was veri®ed. In these experiments, zymosan-

activated plasma (ZAP) was used as chemotactic stim-

ulus, because of the documented ef®cacy of C5a

(generated in plasma by zymosan) to induce profound

tissue accumulation of leucocytes (Arfors et al. 1987).

Intradermal injection of ZAP resulted in a close to 3-

fold increase in leucocyte accumulation (as determined

by MPO activity) in the skin lesions [1.32 � 0.77 units

MPO (g tissue))1] compared with PBS injection

[0.51 � 0.21 units MPO (g tissue))1]. Injection of

prostaglandin E2 (PGE2), a vasodilator which does not

induce leucocyte accumulation per se, yielded MPO

activity [0.45 � 0.32 units MPO (g tissue))1] similar to

that for saline injection. However, the leucocyte accu-

mulation in the skin lesions in response to ZAP stim-

ulation was markedly potentiated by co-administration

of PGE2, as re¯ected by an increase in MPO activity to

2.72 � 0.43 units MPO (g tissue))1 (Fig. 5).

DISCUSSION

Our ®ndings demonstrating that chemoattractant-

induced leucocyte accumulation in rat skin is markedly

enhanced when a potent vasodilator is co-administered

with the chemotactic stimulus con®rm previous ob-

servations that tissue blood ¯ow is an important pa-

rameter in determining leucocyte recruitment to

in¯amed tissue sites (Issekutz & Movat 1982, Downey

et al. 1988, Raud et al. 1988, Teixeira et al. 1993). The

present study was undertaken to investigate by which

means the microvascular blood ¯ow level may in¯uence

the leucocyte extravasation process in in¯ammation.

Intravital microscopic observations of microvessels in

the rat mesentery demonstrated a close relationship

Figure 3 Relationship between rolling leucocyte ¯ux in rat mesen-

teric venules prior to chemotactic stimulation and number of adherent

leucocytes in the same venules following challenge with fMLP

(10±6M, 6 min). Linear regression line is superimposed.

Figure 4 Average rolling velocity of leucocytes which came to arrest

(adherent) within de®ned venular segments in the rat mesentery in

response to fMLP (10)6M) stimulation, and of leucocytes which did

not adhere (non-adherent) in the same vessel segments. Data are

means � SD based on analysis of totally 172 leucocytes. P < 0.05.

Figure 5 Leucocyte accumulation, as measured by MPO activity, in

rat skin after local injection of PBS, PGE2, ZAP (activated comple-

ment factors), or ZAP plus PGE2. Mean � SD, n � 4, P < 0.05.



between the venular blood ¯ow and the rolling ¯ux of

leucocytes. Moreover, there was a strict proportionality

between the chemoattractant-induced ®rm adhesive

response and the rolling cell number, as previously

documented (Lindbom et al. 1992). Consequently, there

is a direct association between the venular blood ¯ow

and the extent of ®rm leucocyte adhesion. In line with

this view are our previous ®ndings that topical ad-

ministration of a vasodilator (acetylcholine) to the rat

mesentery increased venular blood ¯ow, rolling leuco-

cyte ¯ux, and chemoattractant-induced ®rm adhesion

to approximately the same extent (Thorlacius et al.

1995). In an analysis of relationships between small

venular haemodynamics and leucocyte/endothelium

interactions, and their signi®cance for leucocyte re-

cruitment to tissues in in¯ammation, it is important to

stress the fact that variations in tissue blood ¯ow (e.g.

the hyperaemia in in¯ammation) are effectuated

through calibre changes of the feeding arterioles. At the

post-capillary venular level, the corresponding ¯ow

variation will be re¯ected by a change in ¯ow velocity

as these small-sized venules lack the potential of vary-

ing their lumen to any appreciable extent. Conse-

quently, in a given venule, variations in the volume

blood ¯ow will result in proportional changes in blood

¯ow velocity as well as in wall shear stress.

Leucocyte recruitment to extravascular tissue is a

key component in in¯ammatory reactions. In order for

the leucocytes to arrest and establish ®rm contact with

the vascular endothelium in the in¯amed tissue area,

they need to resist the mechanical stress exerted by the

blood stream. To this end, initial rolling along the en-

dothelial lining is a necessary precondition for the ®rm

attachment to occur at physiological blood ¯ow rates

(Lindbom et al. 1992). The rolling interaction, mediated

predominantly by the selectin family of adhesion mol-

ecules (Tedder et al. 1995), serves to retard the motion

of the leucocytes, thereby enabling their ®rm binding to

the vessel wall, which in turn critically depends on the

function of b2 integrins (CD11/CD18) (Arfors et al.

1987). While selectins are capable of supporting a labile

adhesive interaction over a wide range of ¯uid shear,

formation of adhesion bonds via b2 integrins appears

to be markedly shear sensitive. However, once b2

integrin binding (and ®rm cell attachment) is estab-

lished, it becomes shear resistant (Lawrence & Springer

1991). In line with a capacity of the selectins to sustain

leucocyte rolling at high blood ¯ow rates, we found

that neither leucocyte rolling fraction nor the rolling

velocity was quantitatively related to the wall shear rate

in the microvessels. One factor contributing to the

comparably high shear insensitivity of the rolling in-

teraction may be ascribed to the deformability of the

leucocyte in relation to the shear stress imposed. The

rolling leucocytes assume a more ¯attened form with

increasing wall shear rate, and thereby, the contact area

(and adhesion strength) between the leucocyte and the

endothelial surface supposedly will increase (Firrell &

Lipowsky 1989; Damiano et al. 1996).

The apparent insensitivity of the rolling interaction

to varying shear conditions indicated by the present

observations differs from previously reported data on

various degree of inverse proportionality between shear

determinants and rolling parameters in microvessels

in vivo (Firrell & Lipowsky 1989, Ley & Gaehtgens

1991, Perry & Granger 1991, Damiano et al. 1996) and

in ¯ow chamber systems in vitro (Lawrence et al. 1987,

Lawrence & Springer 1991). The reason for these dis-

crepancies may, with respect to the in vivo studies,

pertain to differences in the size of vessels in which

observations have been made. In the present investi-

gation, the category of small-sized venules (15±25 lm)

where the most prominent leucocyte response to che-

moattractant stimulation normally takes place was

chosen for study. Also, in some of the studies quoted,

wall shear rate was lowered well below the normally

prevailing levels by forced reductions in blood ¯ow,

which may have in¯uenced the relationships involved.

The same problem applies to ®ndings obtained in dif-

ferent in vitro models, as the shear stress levels at which

observations typically have been made are more than

one order of magnitude lower than those present at

physiological ¯ow rates in vivo.

Our ®nding that leucocyte rolling velocity varied

within a rather narrow range and showed no correlation

to wall shear rate, suggests that rolling velocity princi-

pally is determined by the rate of formation and

breakage of the adhesive bonds (ToÈzeren & Ley 1992,

Alon et al. 1995) rather than being a function of the

¯uid drag force. However, this conclusion rests on the

fact that we considered only steady rolling leucocytes in

the analysis of rolling velocity and not those which were

intermittently rolling (jumping) along the endothelial

lining. Inasmuch as the ¯ow rate will affect transit time

of intermittently rolling cells similarly as for the free-

¯owing cells, a less restrictive criterion than here used

to delimit the rolling cell population likely accounts for

a seemingly discordant view with regard to the in¯u-

ence of wall shear rate on mean leucocyte rolling

velocity (Ley & Gaehtgens 1991).

We have previously shown that ®rm leucocyte ad-

hesion induced by chemoattractants is directly pro-

portional to the rolling cell number (Lindbom et al.

1992). The ®ndings of the present study extend these

observations to include a dependency of ®rm adhesion

also on the rolling velocity. We found that the average

rolling velocity of those leucocytes which upon

chemotactic stimulation ®rmly adhered in the observed

venular segments was signi®cantly lower than the ve-

locity of those cells which did not arrest but continued



to roll in the same vessel segments. In fact, rolling

velocities in the range of 25 lm s)1 and above were

exceptional among those leucocytes which came to

arrest. The existence of a critical rolling velocity level

above which transition into ®rm adhesion rarely will

take place is consistent with the shear sensitivity of û2

integrins in establishing ®rm binding to the vessel wall

(Worthen et al. 1987, Lawrence & Springer 1991,

Lindbom et al. 1992), and conform with previously

described relationships for spontaneously adhering

leucocytes in the cat mesentery (Perry & Granger 1991).

Even though there exists no clear relationship between

leucocyte rolling and venular shear forces at physio-

logical blood ¯ow rates, profound reductions in blood

¯ow (and shear rate) will ultimately lead to a decrease in

rolling velocity. Consequently, at very low ¯ow rates,

such as in ischaemic conditions, ®rm leucocyte adhe-

sion may be facilitated because of low rolling velocities

present in that situation. Such relationship may account

for the increased spontaneous adhesion found with

decreasing venular wall shear rate associated with

forced reductions in blood ¯ow (Perry & Granger

1991).

Taken together, we have demonstrated that there

exists a quantitative relationship between venular blood

¯ow and the extent of leucocyte rolling on the one

hand, and between leucocyte rolling and the ®rm ad-

hesive response induced by chemoattractants on the

other. Thus, through these relationships, a link between

tissue hyperaemia and leucocyte recruitment, charac-

teristic features of the in¯ammatory response, is pro-

vided. The in¯uence in this process of venular shear

forces on the binding dynamics of the adhesion mole-

cules involved appears to be of minor importance, in-

asmuch as no association between wall shear rate and

the rolling interaction or the ®rm adhesive response

was indicated by the data.

This study was supported by the Swedish Medical Research Council

(14X-4342, 04P-10738), the Swedish Foundation for Health Care

Sciences and Allergy Research (A98110), IngaBritt and Arne Lund-

bergs Foundation, and Karolinska Institutet.

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