Spontaneous leukocyte rolling in venules in untraumatized skin of conscious and anesthetized animals

1994 ◽  
Vol 267 (3) ◽  
pp. H1199-H1204 ◽  
Author(s):  
G. H. Janssen ◽  
G. J. Tangelder ◽  
M. G. Oude Egbrink ◽  
R. S. Reneman
Keyword(s):  
Defense Mechanisms ◽  
Fluorescent Labeling ◽  
Leukocyte Rolling ◽  
Rolling Velocity ◽  
Conscious Rats ◽  
Swiss Mice ◽  
Anesthetized Rats ◽  
Mouse Ear ◽  
Rats And Mice

Intravital bright-field videomicroscopy was used to investigate whether leukocyte rolling can be observed under normal physiological conditions. We studied skin venules in trained conscious rats and anesthetized rats and mice without touching the skin itself. Leukocyte rolling was spontaneously present in all hindpaw venules of Lewis rats (diam 8-27 microns) and also in all mouse ear venules (Swiss, 13-38 microns; BALB/c, 12-56 microns). Rolling levels (in leukocytes/min, median and range) were 8 (3-15) in conscious rats, 9 (3-19) in anesthetized rats, 30 (5-160) in anesthetized Swiss mice, and 10 (3-22) in anesthetized BALB/c mice. These levels appeared to be independent of time. Noninvasive mechanical stimulation induced an average increase of 32%. Fluorescent labeling of leukocytes in vivo with acridine orange had no influence. In Swiss mice, the rolling velocity was < 50 microns/s for > 75% of the leukocytes (median 31 microns/s); this parameter did not correlate with reduced velocity (17-68 s-1) and hence wall shear rate. Our finding that leukocyte rolling is spontaneously present in skin venules of anesthetized and conscious animals suggests a constant vigilance of the host defense mechanisms in the skin.

scholarly journals Sialyl Lewisx (sLex) and an sLexMimetic, CGP69669A, Disrupt E-Selectin–Dependent Leukocyte Rolling In Vivo

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 475-483 ◽  
Author(s):  
Keith E. Norman ◽  
Gary P. Anderson ◽  
Hartmut C. Kolb ◽  
Klaus Ley ◽  
Beat Ernst
Keyword(s):  
Leukocyte Rolling ◽  
Sialyl Lewisx ◽  
Necrosis Factor Alpha ◽  
Rolling Velocity ◽  
Beneficial Effects ◽  
Selectin Family ◽  
Factor Alpha ◽  
Observable Event

Abstract Leukocyte rolling is the earliest observable event in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewisx (sLex) is required for function. Despite findings that sLex inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin–dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNFα)-stimulated mouse cremaster is increased following treatment with either sLex or the sLex-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti–P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin–dependent) rolling is unaffected by either sLex or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.

scholarly journals Leukocyte recruitment in the airways: an intravital microscopic study of rat tracheal microcirculation

2002 ◽  
Vol 282 (5) ◽  
pp. L959-L967 ◽  
Author(s):  
Lina H. K. Lim ◽  
Bruce S. Bochner ◽  
Elizabeth M. Wagner
Keyword(s):  
Real Time ◽  
Leukocyte Recruitment ◽  
New Method ◽  
Histological Evaluation ◽  
Leukocyte Rolling ◽  
Adherent Cells ◽  
Rolling Velocity ◽  
Leukocyte Extravasation ◽  
Local Superfusion

Because of its relative inaccessibility, inflammatory cell extravasation within the airway circulation in vivo has been difficult to investigate in real time. A new method has been established using intravital microscopy in the anesthetized rat to visualize leukocytes in superficial postcapillary venules of the trachea. This technique has been validated using local superfusion of lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Basal leukocyte rolling velocity (55.4 ± 9.3 μm/s) and adhesion (1.4 ± 0.3 cells/100 μm) were monitored in postcapillary venules (33.9 ± 1.3 μm diameter). At all time points up to 90 min, these parameters were unaltered in control rats ( n= 7). In contrast, vessels exposed to 1 μg/ml of LPS ( n = 6) exhibited a 57% reduction in leukocyte rolling velocity and an increase in the number of adherent cells (4.7 ± 1 cells/100 μm, P < 0.05). Superfusion with 0.1 μM of FMLP ( n = 6) also resulted in a 45% reduction in rolling velocity and an increase in adherent cells (4 ± 0.7 cells/100 μm, P < 0.05). Histological evaluation confirmed local stimulus-induced leukocyte extravasation. These results demonstrate leukocyte recruitment in the airway microvasculature and provide an important new method to study airway inflammation in real time.

Molecular mechanisms involved in superoxide-induced leukocyte-endothelial cell interactions in vivo

1994 ◽  
Vol 266 (2) ◽  
pp. H637-H642 ◽  
Author(s):  
J. P. Gaboury ◽  
D. C. Anderson ◽  
P. Kubes
Keyword(s):  
Molecular Mechanisms ◽  
Leukocyte Adhesion ◽  
Platelet Activating Factor ◽  
Leukocyte Rolling ◽  
Rolling Velocity ◽  
Paf Receptor ◽  
Rolling Leukocytes ◽  
Adherent Leukocytes ◽  
Web 2086

Intravital microscopy was used to monitor leukocyte adherence, flux, rolling velocity, and number of rolling leukocytes (flux/velocity) in venules 25–40 microns in diameter. The superoxide-generating system, hypoxanthine and xanthine oxidase (HX/XO), was infused into the mesenteric circulation in untreated animals or in animals pretreated with either catalase (a hydrogen peroxide scavenger), WEB-2086 [a platelet-activating factor (PAF) receptor antagonist], or monoclonal antibodies directed against adhesion molecules CD18 (CL26) or P-selectin (PB1.3). HX/XO infusion caused a decrease in leukocyte rolling velocity and an increase in the number of rolling and adherent leukocytes. WEB-2086 prevented the increase in leukocyte adhesion and markedly increased leukocyte rolling velocity. PB1.3 abolished the HX/XO-associated rise in the flux of rolling leukocytes and proportionally decreased the number of adherent leukocytes. CL26 abolished HX/XO-induced leukocyte adhesion and also reduced the number of rolling leukocytes. In conclusion, P-selectin mediates the increased leukocyte flux induced by superoxide, whereas PAF and CD18 modulate leukocyte adhesion. PAF also reduces leukocyte rolling velocity, possibly as a result of CD18, but not P-selectin.

scholarly journals P-selectin mediates spontaneous leukocyte rolling in vivo

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1308-1316 ◽  
Author(s):  
M Dore ◽  
RJ Korthuis ◽  
DN Granger ◽  
ML Entman ◽  
CW Smith
Keyword(s):  
Intravital Microscopy ◽  
Initial Step ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leukocyte Rolling ◽  
Rolling Velocity ◽  
Inhibition Control ◽  
First Time ◽  
Rolling Leukocytes

Rolling represents the initial step leading to leukocyte extravasation from blood vessels during an inflammatory reaction. In vitro studies indicate that P-selectin could be one of the ligands on endothelium involved in the rolling phenomenon, although the molecular determinants responsible for this transient attachment in vivo are still undefined. Our objectives were to develop a blocking monoclonal antibody against canine P-selectin and to use it to investigate the role of P-selectin in leukocyte rolling in vivo using the technique of intravital microscopy. P-selectin was immunoaffinity purified from canine platelets and used for the production of monoclonal antibodies. One of the hybridomas generated, MD6, was shown by enzyme-linked immunosorbent assay and by flow cytometry to bind preferentially to stimulated platelets and to completely prevent binding of stimulated platelets to neutrophils. Visualization of canine mesenteric venules by intravital microscopy showed that administration of MD6 resulted in a marked inhibition in the number of rolling leukocytes (18.96 +/- 9.92 v 156.40 +/- 19.50 leukocytes/min, P < .05; 88.3% +/- 6.0% inhibition). Control antibody MD3 (which recognizes a nonfunctional epitope of canine P- selectin) had no effect on the number of rolling leukocytes or on their rolling velocity. These results show for the first time that P-selectin plays an essential role in leukocyte rolling in vivo, and therefore may be a key participant of the inflammatory response.

scholarly journals Relevance of L-selectin Shedding for Leukocyte Rolling In Vivo

1999 ◽  
Vol 189 (6) ◽  
pp. 939-948 ◽  
Author(s):  
Ali Hafezi-Moghadam ◽  
Klaus Ley
Keyword(s):  
Leukocyte Rolling ◽  
Rolling Velocity ◽  
Tnf Α ◽  
Novel Method ◽  
Metalloprotease Inhibitor ◽  
Rolling Leukocytes ◽  
Deficient Mice ◽  
Necrosis Factor

The velocity of rolling leukocytes is thought to be determined by the expression of adhesion molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in L-selectin shedding. The hydroxamic acid–based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 μm/s in vivo. When E-selectin was expressed on the endothelium (tumor necrosis factor [TNF]-α treatment 2.5–3 h before the experiment), rolling velocity was 4 μm/s and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 μm/s in E-selectin–deficient mice treated with TNF-α. The reduction of velocity caused by KD-IX-73-4 was immediate (&lt;5 s) after injection of KD-IX-73-4 as shown by a novel method using a local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.

Role of shear forces and adhesion molecule distribution on P-selectin-mediated leukocyte rolling in postcapillary venules

2004 ◽  
Vol 287 (6) ◽  
pp. H2705-H2711 ◽  
Author(s):  
Michael B. Kim ◽  
Ingrid H. Sarelius
Keyword(s):  
Adhesion Molecule ◽  
Expression Patterns ◽  
Local Variation ◽  
Early Event ◽  
High Temporal Resolution ◽  
Leukocyte Rolling ◽  
Shear Forces ◽  
Rolling Velocity ◽  
Local Shear

Rolling on the venular endothelium is a critical step in the recruitment of leukocytes during the inflammatory response. P-selectin is a key mediator of leukocyte rolling, which is an early event in the inflammatory cascade; this rolling is likely to be directly regulated by both local fluid shear forces and P-selectin site densities in the microvasculature. However, neither the spatial pattern of P-selectin expression in postcapillary venules nor the effect of local expression patterns on rolling behavior in intact functional venules is known. We investigated the influence of local shear forces and the spatial distribution of endothelial P-selectin in intact blood perfused post capillary venules in anesthetized mice using intravital confocal microscopy, high temporal resolution particle tracking, and immunofluorescent labeling. We demonstrated a shear-dependent increase in average leukocyte rolling velocity that was attributable to a shear-dependent increase in the occurrence of transient leukocyte detachments from the endothelial surface: translational velocity during leukocyte contact with the vessel wall remained constant. P-selectin expression was not different in venules with characteristically different shear rates or diameters but varied significantly within individual venules. In postcapillary venules, regions of high P-selectin expression correlated with regions of slow leukocyte rolling. Thus the characteristically variable leukocyte rolling in vivo is a function of the spatial heterogeneity in P-selectin expression. The study shows how the local hydrodynamic forces and the nonuniform pattern of P-selectin expression affect the behavior of interacting leukocytes, providing direct evidence for the local variation of adhesion molecule expression as a mechanism for the regulation of leukocyte recruitment.

scholarly journals Sialyl Lewisx (sLex) and an sLexMimetic, CGP69669A, Disrupt E-Selectin–Dependent Leukocyte Rolling In Vivo

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 475-483 ◽  
Author(s):  
Keith E. Norman ◽  
Gary P. Anderson ◽  
Hartmut C. Kolb ◽  
Klaus Ley ◽  
Beat Ernst
Keyword(s):  
Leukocyte Rolling ◽  
Sialyl Lewisx ◽  
Necrosis Factor Alpha ◽  
Rolling Velocity ◽  
Beneficial Effects ◽  
Selectin Family ◽  
Factor Alpha ◽  
Observable Event

Leukocyte rolling is the earliest observable event in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewisx (sLex) is required for function. Despite findings that sLex inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin–dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNFα)-stimulated mouse cremaster is increased following treatment with either sLex or the sLex-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti–P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin–dependent) rolling is unaffected by either sLex or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.

scholarly journals Acute activation of the calcium-sensing receptor inhibits plasma renin activity in vivo

2010 ◽  
Vol 299 (4) ◽  
pp. R1020-R1026 ◽  
Author(s):  
Douglas K. Atchison ◽  
M. Cecilia Ortiz-Capisano ◽  
William H. Beierwaltes
Keyword(s):  
Parathyroid Hormone ◽  
Plasma Renin Activity ◽  
Plasma Renin ◽  
Renin Activity ◽  
Juxtaglomerular Cells ◽  
Calcium Sensing Receptor ◽  
Conscious Rats ◽  
Calcium Sensing ◽  
Anesthetized Rats

In vitro, the renin-secreting juxtaglomerular cells express the calcium-sensing receptor, and its activation with the calcimimetic cinacalcet inhibits renin release. To test whether the activation of calcium-sensing receptor similarly inhibits plasma renin activity (PRA) in vivo , we hypothesized that the calcium-sensing receptor is expressed in juxtaglomerular cells in vivo, and acutely administered cinacalcet would inhibit renin activity in anesthetized rats. Since cinacalcet inhibits parathyroid hormone, which may stimulate renin activity, we sought to determine whether cinacalcet inhibits renin activity by decreasing parathyroid hormone. Lastly, we hypothesized that chronically administered cinacalcet would inhibit basal and stimulated renin in conscious rats. Calcium-sensing receptors and renin were localized in the same juxtaglomerular cells using immunofluorescence in rat cortical slices fixed in vivo. Cinacalcet was administered acutely via intravenous bolus in anesthetized rats and chronically in conscious rats by oral gavage. Acute administration of cinacalcet decreased basal renin activity from 13.6 ± 2.4 to 6.1 ± 1.1 ng ANG I·ml−1·h−1 ( P < 0.001). Likewise, cinacalcet decreased furosemide-stimulated renin from 30.6 ± 2.3 to 21.3 ± 2.3 ng ANG I·ml−1·h−1 ( P < 0.001). In parathyroidectomized rats, cinacalcet decreased renin activity from 9.3 ± 1.3 to 5.2 ± 0.5 ng ANG I·ml−1·h−1 ( P < 0.05) similar to sham-operated controls (13.5 ± 2.2 to 6.6 ± 0.8 ng ANG I·ml−1·h−1, P < 0.05). Chronic administration of cinacalcet over 7 days had no significant effect on PRA under basal or stimulated conditions. In conclusion, calcium-sensing receptors are expressed in juxtaglomerular cells in vivo, and acute activation of these receptors with cinacalcet inhibits PRA in anesthetized rats, independent of parathyroid hormone.

Hydrocortisone reduced in vivo, inflammation-induced slow rolling of leukocytes and their extravasation into human conjunctiva

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2203-2207 ◽  
Author(s):  
Juha Kirveskari ◽  
Maaret Helintö ◽  
Jukka A. O. Moilanen ◽  
Timo Paavonen ◽  
Timo M. T. Tervo ◽  
...  
Keyword(s):  
Human Subjects ◽  
Leukocyte Rolling ◽  
Endothelial Lining ◽  
Rolling Velocity ◽  
Microscopy Technique ◽  
In Vivo Inflammation ◽  
Reflected Light ◽  
Human Conjunctiva ◽  
Conjunctival Vessels

Abstract Hydrocortisone reduces the number of inflammatory leukocytes within tissues, but thus far the site of action on the multistep adhesion cascade leading to leukocyte extravasation has not been identified. We have recently developed a noninvasive in vivo reflected-light confocal microscopy technique to study this at sites of inflammation in human patients. In the present study, we evaluated the effect of preoperative intravenous hydrocortisone treatment on leukocyte trafficking after conjunctival inflammation induced by cataract surgery in human subjects in vivo. The surgery generated leukocyte rolling along the endothelial lining of conjunctival vessels. While preoperative hydrocortisone did not reduce the number of rolling cells, it significantly raised the velocity of individual rolling leukocytes and concomitantly reduced leukocyte emigration into conjunctival tissue. Immunohistology of conjunctival biopsies excised from the individuals studied provided circumstantial evidence that endothelial P-selectin might play a role in the surgery-induced up-regulation of the leukocyte rolling. Furthermore, hydrocortisone reduced surgery-induced P-selectin induction, suggesting a role for this selectin in the regulation of local leukocyte traffic into sites of inflammation in human conjunctiva. Taken together, these results suggest that control of the rolling velocity might be an effective way to adjust leukocyte traffic in vivo in human subjects.

Velocity differences between L- and P-selectin-dependent neutrophil rolling in venules of mouse cremaster muscle in vivo

1996 ◽  
Vol 271 (6) ◽  
pp. H2740-H2747 ◽  
Author(s):  
U. Jung ◽  
D. C. Bullard ◽  
T. F. Tedder ◽  
K. Ley
Keyword(s):  
Intravital Microscopy ◽  
Surgical Trauma ◽  
Leukocyte Rolling ◽  
Cremaster Muscle ◽  
Wild Type ◽  
Rolling Velocity ◽  
Physiological Conditions ◽  
Whole Mount ◽  
Deficient Mice

After surgical trauma, leukocyte rolling is initially normal in L-selectin-deficient mice and reduced at later times, whereas leukocyte rolling is initially absent in P-selectin-deficient mice but induced later. Here, we examined the possibility that P- and L-selectin support rolling at different characteristic velocities using intravital microscopy of venules of the exteriorized cremaster muscle venules of wild type (WT) and P- and L-selectin-deficient mice. At > 50 min after exteriorization, rolling in P-selectin-deficient mice occurred at significantly higher velocities (129 +/- 89 microns/s) than in WT mice (49 +/- 23 microns/s). Rolling velocity distribution in L-selectin-deficient mice was similar to WT mice immediately after exteriorization. Histological examination of Giemsa-stained whole-mount preparations in cremaster muscle venules revealed that the majority of rolling cells (approximately 90% in all genotypes) were granulocytes. We conclude that P-selectin mediates leukocyte rolling at velocities < 50 microns/s, whereas L-selectin sustains more rapid rolling. Under physiological conditions, P- and L-selectin synergize to support rolling at velocities between 20 and 70 microns/s as seen in WT mice.

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