Acta Physiol Scand 1992, 146, 415-421
Chemoattractant-induced firm adhesion of leukocytes t o vascular endothelium in vivo is critically dependent on initial leukocyte rolling L. LINDBOM, X. XIE, J. R A U D and P. HEDQVIST Department of Physiology I, Karolinska Institute, S-104 01 Stockholm, Sweden LINDBOM, L., XIE, x., RAUD,J. & HEDQVIST, P. 1992. Chemoattractant-induced firm adhesion of leukocytes to vascular endothelium in vivo is critically dependent on initial leukocyte rolling. Acta Physiol Scand 146, 415-421. Received 8 July 1992, accepted 29 July 1992. ISSN 0001-6772. Department of Physiology I, Karolinska Institute, Stockholm, Sweden Leukocyte rolling and firm adhesion at the venular endothelium are two discrete events in the cellular inflammatory response mediated via selectin and integrin adhesion molecules, respectively. The dependency of chemoattractant-induced firm leukocyte adhesion on the preceding rolling interaction was investigated in rat mesenteric microvessels through use of intravital microscopy. Leukocyte rolling was dosedependently inhibited by systemic treatment with the sulphated polysaccharide fucoidin. The firm leukocyte adhesion following stimulation with the chemotactic peptide fMLP was similarly inhibited when fMLP challenge was performed subsequent to inhibition of leukocyte rolling by fucoidin. Thus, based on paired observations in single venules before and after fucoidin treatment, reduced rolling leukocyte flux prior to fMLP challenge was paralleled over a wide range by a proportional decrease in fMLP-induced leukocyte adhesion. The results demonstrate quantitatively a close relationship between the extent of leukocyte rolling and the magnitude of the subsequent firm adhesion response, and, that an initial rolling interaction is a precondition for firm adhesion to occur at physiological blood flow rates in vivo. Key words ; adhesion, inflammation, integrins, leukocytes, leukocyte rolling, microcirculation, selectins, venular endothelium.
Acute inflammation involves local intravascular accumulation of polymorphonuclear leukocytes, followed by their emigration to the extravascular space. In this process, the leukocytes interact with the vessel wall in several sequential steps, one of which includes firm attachment to the endothelial cell lining via leukocytic /32 integrins ( C D l l / C D 1 8 ) (Arfors et al. 1987, Carlos & Harlan 1990). I n tissues prepared for intravital microscopy, a fraction of the circulating leukocytes can be seen rolling along the endothelium of small venules at a considerably lower velocity than free-flowing blood components (Atherton &Born Correspondence : Lennart Lindbom, Department of Physiology I, Karolinska Institute, S-104 01 Stockholm, Sweden.
1972,1973). Such leukocyte rolling appears to be initiated/augmented by mild injury (Clark & Clark 1935, Allison et al. 1955, Atherton & Born 1972, Fiebig et al. 1991), and in inflammation rolling represents the earliest established interaction with the venular endothelium, apparently preceding firm attachment of leukocytes upon activation by chemotactic agents. In contrast to the firm adhesion, leukocyte rolling is unaffected by treatment with monoclonal antibodies against the common /32 subunit of CD11/CD18 molecules, suggesting different molecular mechanisms behind the two phenomena (Arfors et al. 1987, Lindbom et al. 1990). I n fact, recent intravital microscopic observations suggest that leukocyte rolling is mediated by cell surface molecules of the selectin family, i.e. leukocyte rolling is largely inhibited
415 15
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b!- interference with leukocytic L-selectin (LEC-+ILI-I, LAM-1, gp!ill''''''jfunction (Le!ill. 1091b, von .lndrian et ( I / . 1991). Although the rolling interaction has long been assumed to be inrolved in the leukocyte emigration process during inflammation, the significance of this phenomenon for t h e firm adhesive response has not been documented in cico. Yet, the possibility that leukoc!-te rolling may b e necessary for firm adhesion via /32 integrins is indicated by in z i t y o data ( L e y et rtl. 1989, Lawrence gi Springer 1991) and by qualitative observations of spontaneous lcukocyte adhesion in Z'IS'Q (von Andrian et u l . 1991). S u c h a relationship is also in line with in z k o findings that neutrophil cxtravasation into inflammatory sites can be inhibited by blocking the function of either CDll/CDl8 (-Arfors et ul. 1987, Price et ul. 1987, L i n d b o m et a / . 1990) or L-selectin (Jutila e t a / . 1989, 1991, Watson et ul. 1991). In the present intravital microscopic stud!- we describe quantitatively the functional relationship between initial leukocj-te rolling and subsequent chemoattractant-induced firm adhesion in r i w .
et
3I;lT E R I AdL S AND 3II E T HOD S .idult female Wstar rats weighing approximately 2.50 g were anaesthetized with equal parts of fluanison/ Janssen fentanyl (Hypnorm, 10/0.2 mg ml-'; Pharmaceutica, Beers, Belgium) and midazolam ; Hoffman-La Roche, Basel, (Dormicum, 5 mg ml ~' Switzerland) diluted 1 : 1 with sterile water (0.2 ml 100 g bod! nt-' intramuscularl!-). T h e trachea was cannulated to facilitate spontaneous breathing. Catheters were placed in the left femoral 1-ein for i.\-. administration of test substances and supplementar!doses of anaesthesia, and in the left femoral artery for blood pressure recording and biood sample collection. Body temperature was maintained at 37 "C by a heating pad connected to a rectal thermistor. Laparotomy was performed by a midline incision, and a segment of the ileum was exteriorized from the peritoneal cavitv and placed on a heated transparent pedestal to allow microscopic observation of the mesenteric microcirculation. The exposed tissue was superfused with a thermostated (37 "C) bicarbonatebuffered saline solution (composition in mht: NaCI 132, KCl t.i, CaC12 2.0, 41gS0, 1.2, NaHCO, 18) equilibrated with soo C 0 2 in nitrogen to maintain physiological pH. Observations of the mesenteric microcirculation were made using a Leitz Orthoplan microscope equipped with water immersion lenses ( x 2.5,X.A 0.6; x .5.5? N A 0.8). The microscopicimage was telex ised (Panasonic Wi-- 1 5.50. COHU 4400 S I T )
and recorded on video tape (Panasonic NV-Fl00 S-C-HS) for subsequent off-line analysis. iifter positioning under the microscope, a 30-min equilibration period preceded quantitative measurements. Analysis of leukocyte-endothelial interactions (rolling and adhesion) was made in small venules (inner diameter 1&20pm) with stable blood flow. The ' rolling leukocyte flux ' was determined at various time intervals by counting the number of rolling leukocytes per min passing a reference point in the microvessel. Leukocyte adhesion was induced by the chemotactic peptide N-formyl-methionyl-leucylphenylalanin ( W L P ; Sigma, St Louis, MO, USA) added topically- via the superfusion buffer at a final concentration of lo-' M for periods of 6-20 min. At the end of the stimulation period, the number of adherent leukocytes per 100 y m length ofthe observed vessel segment was counted. Repeated applications of i3iLP were separated by pertinent washing periods (buffer only for > 12 min) during which the adherent leukocytes dislodged from the endothelial cell lining. iVith prolonged fMLP stimulation (> 15 min) leukocytes remained attached for a longer time and were also seen to extravasate. I n order to inhibit the leukocyte rolling, animals were treated intravenously ivith fucoidin (Sigma) in doses ranging from 0.2 to 25 mg kg-' body wt. From pilot experiments it was evident that this sulphated fucose-rich polysaccharide exhibited a strong capacity to inhibit rolling when given systemically. Challenge with fMLP was performed before and 10 min after fucoidin treatment, and the adhesion responses obtained were related to the respective prestimulatory rolling leukocyte flux. In five experiments, i M L P was applied repeatedly at various times after fucoidin treatment during a period of up to 2 h with pertinent washing sequences in between. T h e quantification of rolling and adherent leukocytes was made under ordinary light transillumination. In order to detect the free-flowing fraction of leukocytes in the observed vascular segments, short periods of incident fluorescent light (Leitz Ploemopak, filter block 12) were used in combination with a simultaneous i.v. infusion of acridine orange ( 5 mg kg-' body wt). These recordings also permitted, through frame-by-frame analysis, determination of the velocity of individual free-flowing leukocytes. From these data estimates of mean blood flow velocity and wall shear rate were calculated (Ley & Gaehtgens 1991). I n four experiments, venular whole blood flow velocity was reduced to levels in the range of the normal leukocyte rolling velocity by partial occlusion of microvessels with a blunt probe positioned by a micromanipulator. T h e systemic polymorphonuclear and mononuclear leukocyte concentrations were determined from arterial blood samples by differential cell counting in a Biirker chamber.
Leukocyte rolling and firm adhesion Values are presented as meansf SEM. Statistical analysis was performed using linear regression, the Wilcoxon test for paired samples, and the KruskalWallis test for multiple comparisons. RESULTS At the end of the equilibration period, the number of leukocytes rolling spontaneously along the vendar endothelium was 1 3 2 2 cells min-' (range: 2-32, n = 29), constituting 23+4y0of the total leukocyte flux (i.e. rolling plus freeflowing leukocytes) in the observed venules. The rolling leukocyte flux in individual venules remained largely constant when followed for up to 1 h in resting tissue, and only occasional cells adhered spontaneously under these circumstances. Within minutes of topical challenge with fh4LP ( M), rolling leukocytes started to -r - 1
15-
.b Ei
5: 2* ;-
10-
-
0-0
u r ,
+;52 2 2 M O ski
cds
E 4
0 ,
B
,
,
,
,
,
,
,
,
I
1 0.2
Fig. 2. Effect of intravenous treatment with fucoidin on flux of spontaneously rolling leukocytes. Mean vaIues$SEM (three animals in each group) as a percentage of the rolling flux value before treatment with fucoidin (or saline, bar on far left).
417
adhere to the venular endothelium, conconiitantly with a decrease of rolling leukocyte flux (Fig. 1). In response to prolonged stimulation (20 min), the number of adherent leukocytes began to plateau after approximately 10 min, seemingly reaching a maximal response to the particular dose chosen (Fig. I). It also became evident that leukocytes which adhered firmly to the vessel wall always rolled along the endothelium for some distance before they attached, i.e. the adherent leukocytes were recruited from the rolling cell population exclusively. Upon withdrawal of the chemoattractant, the adherent cells either dislodged from the endothelium and were swept away by the blood stream (particularly after a short period of stimulation, 6min) or extravasated at the site of adhesion, thus rendering the vessels clear from adherent leukocytes within 10-15 min. A second challenge with fMLP resulted in a response not different from the first one. Other chemotactic agents, leukotriene B, (LTB,) and zymosan-activated plasma, induced adhesion responses with dynamics similar to the fMLP response (not shown). T o investigate the inter-relationship between leukocyte rolling and subsequent firm adhesion, rolling was inhibited by intravenous administration of the sulphated polysaccharide fucoidin. Fucoidin treatment resulted in a dose-dependent reduction in the number of spontaneously rolling leukocytes (Fig. 2), as well as a decrease in their transit time due to increased rolling velocity (not shown). T h e ratio of rolling to free-flowing leukocytes in the observed venules decreased from 0.31 & 0.07 to 0.07,0.04 with fucoidin 5 mg kg-l body wt, while the total leukocyte flux remained unchanged (91 & 10% of control, P = 0.23). The systemic polymorphonuclear cell count was unchanged and that of mononuclear cells slightly but significantly increased by this treatment. In separate experiments, fMLP challenge was combined with fucoidin treatment. The mesenteric tissue was challenged with fMLP ( M for 6 min) before and 10min after treatment with fucoidin. Fucoidin treatment resulted in a dose-dependent decrease in both rolling leukocyte flux, measured prior to stimulation with fMLP, and leukocyte adhesion, measured at the end of the stimulation period. Thus, based on these paired observations in single venules before and after fucoidin treatment, the decrease in 15-2
418
L. Lindbom et al.
Time (min)
tucaidiri img k e '1
Fig. 3. Rolling leukocyte flux (0) and f3lLP-induced (l0Fb 51 for 6 min) leukocyte adhesion (a) after intravenous treatment with fucoidin (or saline, bars labelled '0') as a percentage of the corresponding paired value before treatment (---), Rolling leukocyte flux rcfers to the number of rolling leukocl-tesper min prior t o stimulation with fllLP. Yalues are meanj: SEM of five animals in each group. * P < 0.05; *** P < 0.001, Kruskal-LVallis test.
1
0
5
10
15
20
25
1 30
Roiling leukocyte flux (mu-')
Fig. 4. Relationship between rolling leukocyte flux prior to chemotactic stimulation and number clf adherent leukocytes follow-ing f3ll.P challenge. Data are based on the material presented in Figure 3 and
represent observations before and after fucoidin treatment in 20 individual vessels. I' = 0.59, P
Chemoattractant-induced firm adhesion of leukocytes t o vascular endothelium in vivo is critically dependent on initial leukocyte rolling L. LINDBOM, X. XIE, J. R A U D and P. HEDQVIST Department of Physiology I, Karolinska Institute, S-104 01 Stockholm, Sweden LINDBOM, L., XIE, x., RAUD,J. & HEDQVIST, P. 1992. Chemoattractant-induced firm adhesion of leukocytes to vascular endothelium in vivo is critically dependent on initial leukocyte rolling. Acta Physiol Scand 146, 415-421. Received 8 July 1992, accepted 29 July 1992. ISSN 0001-6772. Department of Physiology I, Karolinska Institute, Stockholm, Sweden Leukocyte rolling and firm adhesion at the venular endothelium are two discrete events in the cellular inflammatory response mediated via selectin and integrin adhesion molecules, respectively. The dependency of chemoattractant-induced firm leukocyte adhesion on the preceding rolling interaction was investigated in rat mesenteric microvessels through use of intravital microscopy. Leukocyte rolling was dosedependently inhibited by systemic treatment with the sulphated polysaccharide fucoidin. The firm leukocyte adhesion following stimulation with the chemotactic peptide fMLP was similarly inhibited when fMLP challenge was performed subsequent to inhibition of leukocyte rolling by fucoidin. Thus, based on paired observations in single venules before and after fucoidin treatment, reduced rolling leukocyte flux prior to fMLP challenge was paralleled over a wide range by a proportional decrease in fMLP-induced leukocyte adhesion. The results demonstrate quantitatively a close relationship between the extent of leukocyte rolling and the magnitude of the subsequent firm adhesion response, and, that an initial rolling interaction is a precondition for firm adhesion to occur at physiological blood flow rates in vivo. Key words ; adhesion, inflammation, integrins, leukocytes, leukocyte rolling, microcirculation, selectins, venular endothelium.
Acute inflammation involves local intravascular accumulation of polymorphonuclear leukocytes, followed by their emigration to the extravascular space. In this process, the leukocytes interact with the vessel wall in several sequential steps, one of which includes firm attachment to the endothelial cell lining via leukocytic /32 integrins ( C D l l / C D 1 8 ) (Arfors et al. 1987, Carlos & Harlan 1990). I n tissues prepared for intravital microscopy, a fraction of the circulating leukocytes can be seen rolling along the endothelium of small venules at a considerably lower velocity than free-flowing blood components (Atherton &Born Correspondence : Lennart Lindbom, Department of Physiology I, Karolinska Institute, S-104 01 Stockholm, Sweden.
1972,1973). Such leukocyte rolling appears to be initiated/augmented by mild injury (Clark & Clark 1935, Allison et al. 1955, Atherton & Born 1972, Fiebig et al. 1991), and in inflammation rolling represents the earliest established interaction with the venular endothelium, apparently preceding firm attachment of leukocytes upon activation by chemotactic agents. In contrast to the firm adhesion, leukocyte rolling is unaffected by treatment with monoclonal antibodies against the common /32 subunit of CD11/CD18 molecules, suggesting different molecular mechanisms behind the two phenomena (Arfors et al. 1987, Lindbom et al. 1990). I n fact, recent intravital microscopic observations suggest that leukocyte rolling is mediated by cell surface molecules of the selectin family, i.e. leukocyte rolling is largely inhibited
415 15
ACT 146
116
L. Lindbom et al.
b!- interference with leukocytic L-selectin (LEC-+ILI-I, LAM-1, gp!ill''''''jfunction (Le!ill. 1091b, von .lndrian et ( I / . 1991). Although the rolling interaction has long been assumed to be inrolved in the leukocyte emigration process during inflammation, the significance of this phenomenon for t h e firm adhesive response has not been documented in cico. Yet, the possibility that leukoc!-te rolling may b e necessary for firm adhesion via /32 integrins is indicated by in z i t y o data ( L e y et rtl. 1989, Lawrence gi Springer 1991) and by qualitative observations of spontaneous lcukocyte adhesion in Z'IS'Q (von Andrian et u l . 1991). S u c h a relationship is also in line with in z k o findings that neutrophil cxtravasation into inflammatory sites can be inhibited by blocking the function of either CDll/CDl8 (-Arfors et ul. 1987, Price et ul. 1987, L i n d b o m et a / . 1990) or L-selectin (Jutila e t a / . 1989, 1991, Watson et ul. 1991). In the present intravital microscopic stud!- we describe quantitatively the functional relationship between initial leukocj-te rolling and subsequent chemoattractant-induced firm adhesion in r i w .
et
3I;lT E R I AdL S AND 3II E T HOD S .idult female Wstar rats weighing approximately 2.50 g were anaesthetized with equal parts of fluanison/ Janssen fentanyl (Hypnorm, 10/0.2 mg ml-'; Pharmaceutica, Beers, Belgium) and midazolam ; Hoffman-La Roche, Basel, (Dormicum, 5 mg ml ~' Switzerland) diluted 1 : 1 with sterile water (0.2 ml 100 g bod! nt-' intramuscularl!-). T h e trachea was cannulated to facilitate spontaneous breathing. Catheters were placed in the left femoral 1-ein for i.\-. administration of test substances and supplementar!doses of anaesthesia, and in the left femoral artery for blood pressure recording and biood sample collection. Body temperature was maintained at 37 "C by a heating pad connected to a rectal thermistor. Laparotomy was performed by a midline incision, and a segment of the ileum was exteriorized from the peritoneal cavitv and placed on a heated transparent pedestal to allow microscopic observation of the mesenteric microcirculation. The exposed tissue was superfused with a thermostated (37 "C) bicarbonatebuffered saline solution (composition in mht: NaCI 132, KCl t.i, CaC12 2.0, 41gS0, 1.2, NaHCO, 18) equilibrated with soo C 0 2 in nitrogen to maintain physiological pH. Observations of the mesenteric microcirculation were made using a Leitz Orthoplan microscope equipped with water immersion lenses ( x 2.5,X.A 0.6; x .5.5? N A 0.8). The microscopicimage was telex ised (Panasonic Wi-- 1 5.50. COHU 4400 S I T )
and recorded on video tape (Panasonic NV-Fl00 S-C-HS) for subsequent off-line analysis. iifter positioning under the microscope, a 30-min equilibration period preceded quantitative measurements. Analysis of leukocyte-endothelial interactions (rolling and adhesion) was made in small venules (inner diameter 1&20pm) with stable blood flow. The ' rolling leukocyte flux ' was determined at various time intervals by counting the number of rolling leukocytes per min passing a reference point in the microvessel. Leukocyte adhesion was induced by the chemotactic peptide N-formyl-methionyl-leucylphenylalanin ( W L P ; Sigma, St Louis, MO, USA) added topically- via the superfusion buffer at a final concentration of lo-' M for periods of 6-20 min. At the end of the stimulation period, the number of adherent leukocytes per 100 y m length ofthe observed vessel segment was counted. Repeated applications of i3iLP were separated by pertinent washing periods (buffer only for > 12 min) during which the adherent leukocytes dislodged from the endothelial cell lining. iVith prolonged fMLP stimulation (> 15 min) leukocytes remained attached for a longer time and were also seen to extravasate. I n order to inhibit the leukocyte rolling, animals were treated intravenously ivith fucoidin (Sigma) in doses ranging from 0.2 to 25 mg kg-' body wt. From pilot experiments it was evident that this sulphated fucose-rich polysaccharide exhibited a strong capacity to inhibit rolling when given systemically. Challenge with fMLP was performed before and 10 min after fucoidin treatment, and the adhesion responses obtained were related to the respective prestimulatory rolling leukocyte flux. In five experiments, i M L P was applied repeatedly at various times after fucoidin treatment during a period of up to 2 h with pertinent washing sequences in between. T h e quantification of rolling and adherent leukocytes was made under ordinary light transillumination. In order to detect the free-flowing fraction of leukocytes in the observed vascular segments, short periods of incident fluorescent light (Leitz Ploemopak, filter block 12) were used in combination with a simultaneous i.v. infusion of acridine orange ( 5 mg kg-' body wt). These recordings also permitted, through frame-by-frame analysis, determination of the velocity of individual free-flowing leukocytes. From these data estimates of mean blood flow velocity and wall shear rate were calculated (Ley & Gaehtgens 1991). I n four experiments, venular whole blood flow velocity was reduced to levels in the range of the normal leukocyte rolling velocity by partial occlusion of microvessels with a blunt probe positioned by a micromanipulator. T h e systemic polymorphonuclear and mononuclear leukocyte concentrations were determined from arterial blood samples by differential cell counting in a Biirker chamber.
Leukocyte rolling and firm adhesion Values are presented as meansf SEM. Statistical analysis was performed using linear regression, the Wilcoxon test for paired samples, and the KruskalWallis test for multiple comparisons. RESULTS At the end of the equilibration period, the number of leukocytes rolling spontaneously along the vendar endothelium was 1 3 2 2 cells min-' (range: 2-32, n = 29), constituting 23+4y0of the total leukocyte flux (i.e. rolling plus freeflowing leukocytes) in the observed venules. The rolling leukocyte flux in individual venules remained largely constant when followed for up to 1 h in resting tissue, and only occasional cells adhered spontaneously under these circumstances. Within minutes of topical challenge with fh4LP ( M), rolling leukocytes started to -r - 1
15-
.b Ei
5: 2* ;-
10-
-
0-0
u r ,
+;52 2 2 M O ski
cds
E 4
0 ,
B
,
,
,
,
,
,
,
,
I
1 0.2
Fig. 2. Effect of intravenous treatment with fucoidin on flux of spontaneously rolling leukocytes. Mean vaIues$SEM (three animals in each group) as a percentage of the rolling flux value before treatment with fucoidin (or saline, bar on far left).
417
adhere to the venular endothelium, conconiitantly with a decrease of rolling leukocyte flux (Fig. 1). In response to prolonged stimulation (20 min), the number of adherent leukocytes began to plateau after approximately 10 min, seemingly reaching a maximal response to the particular dose chosen (Fig. I). It also became evident that leukocytes which adhered firmly to the vessel wall always rolled along the endothelium for some distance before they attached, i.e. the adherent leukocytes were recruited from the rolling cell population exclusively. Upon withdrawal of the chemoattractant, the adherent cells either dislodged from the endothelium and were swept away by the blood stream (particularly after a short period of stimulation, 6min) or extravasated at the site of adhesion, thus rendering the vessels clear from adherent leukocytes within 10-15 min. A second challenge with fMLP resulted in a response not different from the first one. Other chemotactic agents, leukotriene B, (LTB,) and zymosan-activated plasma, induced adhesion responses with dynamics similar to the fMLP response (not shown). T o investigate the inter-relationship between leukocyte rolling and subsequent firm adhesion, rolling was inhibited by intravenous administration of the sulphated polysaccharide fucoidin. Fucoidin treatment resulted in a dose-dependent reduction in the number of spontaneously rolling leukocytes (Fig. 2), as well as a decrease in their transit time due to increased rolling velocity (not shown). T h e ratio of rolling to free-flowing leukocytes in the observed venules decreased from 0.31 & 0.07 to 0.07,0.04 with fucoidin 5 mg kg-l body wt, while the total leukocyte flux remained unchanged (91 & 10% of control, P = 0.23). The systemic polymorphonuclear cell count was unchanged and that of mononuclear cells slightly but significantly increased by this treatment. In separate experiments, fMLP challenge was combined with fucoidin treatment. The mesenteric tissue was challenged with fMLP ( M for 6 min) before and 10min after treatment with fucoidin. Fucoidin treatment resulted in a dose-dependent decrease in both rolling leukocyte flux, measured prior to stimulation with fMLP, and leukocyte adhesion, measured at the end of the stimulation period. Thus, based on these paired observations in single venules before and after fucoidin treatment, the decrease in 15-2
418
L. Lindbom et al.
Time (min)
tucaidiri img k e '1
Fig. 3. Rolling leukocyte flux (0) and f3lLP-induced (l0Fb 51 for 6 min) leukocyte adhesion (a) after intravenous treatment with fucoidin (or saline, bars labelled '0') as a percentage of the corresponding paired value before treatment (---), Rolling leukocyte flux rcfers to the number of rolling leukocl-tesper min prior t o stimulation with fllLP. Yalues are meanj: SEM of five animals in each group. * P < 0.05; *** P < 0.001, Kruskal-LVallis test.
1
0
5
10
15
20
25
1 30
Roiling leukocyte flux (mu-')
Fig. 4. Relationship between rolling leukocyte flux prior to chemotactic stimulation and number clf adherent leukocytes follow-ing f3ll.P challenge. Data are based on the material presented in Figure 3 and
represent observations before and after fucoidin treatment in 20 individual vessels. I' = 0.59, P
