Search

We investigated the effects of chronic cocaine exposure on the microcirculation in the rat mesenteric venules under both non-inflammatory and FMLP-induced inflammatory conditions. Chronic cocaine significantly increased WBC rolling flux in both conditions, and potentiated FMLP-induced leukocyte-endothelial cell adhesion (LEA). In cocaine-treated animals, total WBC number increased by 91%, and the ratio of white blood cell to red blood cell velocity was significantly lower, while vessel diameter was unchanged. Chronic cocaine decreased serum levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), but had no effect on interleukin-1 beta (IL-1beta). Expression of intercellular adhesion molecule-1 (ICAM-1) was increased in mesenteric venules following chronic cocaine exposure, and may be one of the mechanisms underlying enhancement of FMLP-induced LEA. The increase in WBC count, WBC flux and LEA, and the change in cell velocity seen in the cocaine-treated animals could cause a decrease in effective vessel diameter and a change in intravascular resistance, and may underlie the progressive vascular damage seen in chronic cocaine-abusing individuals.

Circadian rhythms are responsible for a number of key cycles within the body. In vivo microscopy was used to investigate the hypothesis that the circadian rhythm of corticosterone in rats produces different leukocyte-endothelium interactions throughout the day. The data indicate that corticosterone levels range from 12 ng/ml in the AM to 260 ng/ml in the PM. In contrast, the number of circulating polymorphonuclear leukocytes (PMNs) yields peak values in the AM (630 PMNs/microl) and trough values in the PM (262 PMNs/microl). During surgical stress there is a significant increase in the number of circulating PMNs in the PM but little change in the AM. Furthermore, there is significantly greater leukocyte-endothelium adhesion in the PM (5.2 cells/100 microm) than in the AM (2.9 cells/100 microm). Addition of the chemoattractant FMLP increased adhesion 125% in the AM but only 62% in PM. Both exogenous glucocorticoid supplementation for 2 weeks and bilateral adrenalectomy abolished the circadian rhythms of circulating PMNs, the number of sticking white blood cells and the initial stages of an acute inflammatory response. These findings suggest that the circadian rhythm of corticosterone alters leukocyte-endothelium interactions throughout the day.

Techniques of intravital microscopy were used to assess the effect of the nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and ibuprofen, on the microcirculation. Hemodynamics in venules of the rat mesentery were studied in terms of vessel diameter, red blood cell velocity, and leukocyte-endothelium interactions: leukocyte-endothelium adhesion (LEA), white blood cell (WBC) marginating flux, and WBC velocity. Measurements were made during (1) control conditions (topical suffusion with ringer-gelatin drip), (2) topically suffused indomethacin or ibuprofen, (3) an induced inflammatory response (suffusion with the chemoattractant N-Formyl-Methionyl-Leucyl-Phenylalanine (FMLP)), and (4) concomitant suffusion with FMLP and NSAID. Short term topical suffusion (90 sec) with indomethacin and ibuprofen had little or no effect on control hemodynamics. Five-minute suffusions with indomethacin (5 x 10(-5) to 5 x 10(-4) M) significantly increased LEA while ibuprofen (5 x 10(-3) M) significantly decreased LEA. Topical suffusion with the chemotactic agent FMLP induced inflammation and significantly increased LEA in venules. Treatment with indomethacin during induced inflammation had no effect on the inflammatory reaction in terms of the microvascular hemodynamics measured in this study. Treatment with ibuprofen during induced inflammation significantly reduced LEA and increased red blood cell velocity. In conclusion, although both of the NSAIDs studied here are known to block the cyclooxygenase pathway of arachidonic acid metabolism, the actions of indomethacin and ibuprofen on the inflammatory process are very different with an important effect of ibuprofen being to decrease LEA.

In vivo microscopy was used to assess the relative contribution of hydrodynamic forces (network topography and shear rate) and the specificity for leukocytes to interact with venular endothelium as determinants of leukocyte-endothelium interactions. To ascertain this, microvascular networks in the rat and rabbit mesentery were examined under normograde and mechanically induced retrograde flows to determine the effect of reversed flow on leukocyte-endothelium interactions in arterioles and venules. The data indicate that retrograde perfusion under hemodynamic (red blood cell velocity and shear rate) states equivalent to normograde flow significantly increased leukocyte marginating flux in arterioles (from 0 to 0.5 cells/5 sec) and decreased flux significantly in venules (from 1.0 to 0.2 cells/5 sec). The increased flux in arterioles under retrograde conditions, however, was significantly lower than the flux in venules under normograde conditions and the decreased flux in venules during retrograde flow was significantly greater than the flux in arterioles during normograde flow. This apparent discrepancy appears to be the result of a heterogeneous distribution of adhesive receptors on vascular endothelium. Furthermore, marginating leukocytes in arterioles made only brief contact with the endothelium before being swept away while marginating leukocytes in venules during normal and retrograde perfusion rolled along the vascular wall, with similar velocities in both directions. In conclusion, although hydrodynamic forces are important in facilitating leukocyte margination through mechanisms of radial migration, it is leukocyte-endothelium specificity in venules that ultimately determines leukocyte-endothelium interactions.

In vivo dynamics of the interaction between leukocytes and the endothelium following direct activation of the white blood cells (WBCs), apart from possible endothelial cell activation, were studied in arterioles, capillaries, and venules of splanchnic tissue (rabbit omentum). WBCs were isolated using either density gradient or centrifugation techniques, labeled with fluorescent dyes, and exposed to physiological solutions with or without the chemoactivator N-formyl-methionyl-leucyl-phenlyalanine (FMLP). WBCs isolated using standard density gradient separation techniques rapidly disappeared from the circulating pool following a bolus injection and were sequestered in lung microvessels. The centrifugation technique produced cells that circulated for at least 60 min. WBCs directly activated with FMLP adhered to venular endothelium but not to arteriolar endothelium, suggesting that differences in hydrodynamics in the arteriolar and venular network or fundamental differences between arteriolar and venular endothelia may explain the lack of leukocyte-endothelium adhesion (LEA) in arterioles. WBCs pretreated with FMLP had significantly longer attachment times than nontreated cells, 13.4 and 2.5 sec respectively, which may be indicative of specific receptor chemistry. Similarities in the LEA attachment-detachment process for splanchnic tissue with that previously reported for lymphoid tissue suggest that a fundamental process of cell to cell interaction may exist in all tissues.

Quantitative estimates of the force of adhesion between leukocytes and endothelium were obtained from in vivo hemodynamic measurements in small venules of cat mesentery during topical application of the chemotactic compound N-formyl-methionyl-leucyl-phenylalanine (FMLP). Simultaneous measurements of upstream to downstream pressure drop, red cell velocity, microvessel hematocrit, and vessel diameter and length permitted application of the principles of momentum conservation to calculate the forces acting upon a leukocyte during adhesion to the endothelium. For venules ranging in diameter from 23 to 49 micron, the ratio of force (acting in the vessel axial direction) to wall shear stress on the endothelium fell from 14.6 X 10(-6) in small venules to 2.3 X 10(-6) dynes per dyne/cm2 in large venules; reflecting the larger pressure drops and forces attendant to greater lumen obstruction in the smaller venules. The equilibrium force representative of a balance between fluid shear stresses on the leukocyte and those at its site of contact with the endothelium ranged from 1.1 to 76.1 X 10(-5) dynes for wall shear stress ranging from 2 to 25 dynes/cm2; with venules with greater wall shear stresses having the greater leukocyte-endothelium shear force. Within individual venules, however, the force acting on a single leukocyte varied inversely with wall shear stress, most likely due to white blood cell deformation, which leads to a lessening of shear stress on the surface of the white blood cell.

In the present study we have attempted to provide quantitative details of hemodynamic determinants of leukocyte to endothelium adhesion in the microvasculature. To this end, several hypotheses have been advanced to suggest that the preferential adhesion of leukocytes in the larger venules (30-50 microns diameter) rests upon the inherent ability of the microvasculature to compensate for small perturbations in resistance and that WBC deformability may play a significant role in this process. Flow redistribution and attendant arteriolar vasomotor adjustments may forestall LEA in the larger collecting venules of the network, where venous obstruction may be countered by bringing the full weight of the arteriovenous pressure gradient to oppose WBC adhesion. Direct measurements of the force of adhesion suggest that with diminishing vessel diameter, as for example in the immediate post-capillary venules, WBC dispersal forces will be greatest due to dramatic increases in the proportionality between force and wall shear stress. This event would tend to preclude adhesion in the smallest venular microvessels. It has also been shown that there is a strong potential for WBC deformability to affect the adhesion process by modification of the shear stresses acting on the WBC surface, as evidenced by an inverse relationship between force and wall shear stress and direct observations of WBC shape changes with increasing shear.

The effects of leukocyte-endothelium adhesion on microhemodynamics were studied in cat mesentery under control conditions and following tissue suffusion with the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine (FMLP). The results indicate that under normophysiological conditions there is little or no leukocyte-endothelium adhesion in arterioles and venules. Tissue suffusion with FMLP significantly increases the number of adhering leukocytes in venules, but not in arterioles. Analysis of the number of adhering leukocytes (in venules) as a function of wall shear rate suggested that increased adhesion of leukocytes was primarily due to elevated adhesive forces and not the result of decreases in dispersal forces, i.e., wall shear stress. From measurements of upstream to downstream pressure drop, red cell velocity, and microvessel hematocrit in 16 unbranched venules, no significant changes in diameter (mean of 39.9 +/- 7.8 (SD) micron), intravascular pressure gradient (0.59 +/- 0.40 X 10(-2) cm H2O/micron), nor microvessel hematocrit (31.0 +/- 9.8%) occurred in response to FMLP. There were significant decreases in upstream pressure (8%) and estimated bulk flow (28%) as well as significant increases in the number of adhering leukocytes, from 1.5 +/- 2.8 to 11.4 +/- 8.3 cells/100 micron, and vessel resistance (81%). Changes in hemodynamics were found to be more pronounced in venules with small diameters. The observed response to FMLP suggests that changes in hemodynamics during leukocyte-endothelium adhesion can be accounted for by a decrease in the effective diameter due to obstruction of the lumen by WBCs, and that adhesive interactions between WBCs and endothelium are a major determinant of blood flow resistance in the microcirculation.