Soluble P-selectin antagonist mediates rolling velocity and adhesion of leukocytes in acutely inflamed venules.

OBJECTIVE: Leukocyte rolling is recognized as an important event in facilitating the extravasation of leukocytes from the vascular to the interstitial compartment, and is mediated by the selectin family of cell adhesion molecules. The aim of this study was to evaluate and characterize the rolling behavior of leukocytes in a model of acute inflammation using a novel soluble selectin ligand directed against P-selectin. METHODS: Feline mesenteric postcapillary venules were visualized using intravital microscopy prior to and following exposure to leukotriene C4 (LTC4) in animals pretreated with vehicle (saline) and the P-selectin antagonist rPSGL-Ig. RESULTS: A concentration of 500 pM LTC4 induced a threefold and sixfold elevation in leukocyte rolling flux and adhesion, respectively, compared to baseline values (p < 0.05). Administration of rPSGL-Ig had no effect on LTC4-induced leukocyte rolling flux but significantly attenuated the increase in the fraction of rolling leukocytes (p < 0.05). In addition, rPSGL-Ig inhibited the LTC4-induced reductions in leukocyte rolling velocity (p < 0.001). Finally, LTC4-induced leukocyte adhesion in animals pretreated with rPSGL-Ig was reduced by 60%, compared to vehicle-treated animals (p < 0.05). CONCLUSIONS: LTC4 induces leukocyte rolling and adhesion in feline mesenteric venules in a dose-dependent manner. Administration of rPSGL-Ig inhibits LTC4-induced reductions in leukocyte rolling velocity and attenuates the elevation in the fraction of rolling leukocytes produced by LTC4 stimulation. This suggests that rPSGL-Ig may be used to reduce leukocyte rolling and adhesion, and subsequently attenuate tissue injury during inflammation.

P-Selectin-dependent inhibition of thrombosis during venous stasis.

Leukocyte adhesion, transendothelial migration, and stasis are important components in the pathogenesis of deep vein thrombosis. Anesthetized cats were treated with saline, a recombinant soluble form of P-selectin glycoprotein ligand-1 (rPSGL-Ig), or an E- and L-selectin antibody (EL-246) before exposure and occlusion of a jugular vein. After 2 or 6 hours of occlusion, jugular veins were perfused with buffer, fixed, and prepared for scanning electron microscopy. In cats receiving saline, 2 and 6 hours of occlusion produced moderate levels of leukocyte and platelet adhesion and endothelial cell injury. Treatment of cats with rPSGL-Ig or EL-246 had no apparent effect on the magnitude of cell adhesion and endothelial cell injury compared with no treatment. After 6 hours of occlusion, the presence of a mural thrombus in untreated veins was observed and confirmed by scanning electron microscopy. Pretreatment of cats with rPSGL-Ig completely (4.0 mg/kg) or partially (1.0 mg/kg) prevented the occurrence of thrombi in the jugular veins. The reduction in thrombosis by rPSGL-Ig treatment after 6 hours of venous stasis, in the absence of any effect on leukocyte-mediated endothelial cell injury, suggests an antithrombotic mechanism of action for this protein.

Quantification of murine endothelial cell adhesion molecules in solid tumors.

Coordinated adhesive interactions between lymphocyte receptors and endothelial cell adhesion molecules (CAMs) are a prerequisite for effector cell entry into tumor stroma. Whereas the diminished leukocyte-endothelial cell interactions observed in tumor microvessels have been attributed to a reduced expression of endothelial CAMs, there is no quantitative data bearing on this issue. The dual-radiolabeled monoclonal antibody technique was used to quantify constitutive and tumor necrosis factor (TNF)-alpha-induced expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), ICAM-2, P-selectin, E-selectin, and platelet-endothelial cell adhesion molecule 1 (PECAM-1) in different vascular beds of normal (C57Bl/6) and RM-1 tumor-bearing mice. When corrected for endothelial surface area, the constitutive expression of selectins in tumor vessels was higher than that observed in other vascular beds. Both constitutive and induced expression of endothelial CAMs in peripheral vascular beds did not differ between normal and tumor-bearing mice. Within the tumor, the magnitude of the upregulation of P-selectin, ICAM-1, and VCAM-1 after TNF-alpha was similar to that within other vascular beds. E-selectin expression in tumors was refractory to TNF-alpha, whereas PECAM-1 and ICAM-2 expression were significantly reduced. Our findings suggest that the presence of a solid tumor does not influence endothelial CAM expression in other vascular beds and that the higher density of selectins in nonstimulated tumor vessels may promote the recruitment of rolling leukocytes in this tissue.

Expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in acute and chronic inflammation.

The objective of this study was to quantify, in vivo, constitutive and tumor necrosis factor alpha (TNF-alpha)-enhanced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in different tissues from healthy wild-type mice (C57BL/6) as well as interleukin-10 (IL-10)-deficient mice with and without active colitis. Using the dual radiolabel monoclonal antibody technique, we found substantial constitutive expression of MAdCAM-1 in the intestine, colon, and mesenteric lymph nodes. MAdCAM-1 expression in these tissues was significantly enhanced, in a time-dependent manner, by systemic administration of TNF-alpha. Maximum surface expression was observed at 18 h after TNF-alpha administration and remained significantly elevated at 48 h post-TNF-alpha injection. No significant constitutive nor TNF-alpha-induced expression of MAdCAM-1 was detected in skeletal muscle, brain, or heart. In IL-10-deficient (IL-10 k/o) mice with no clinical or histological evidence of colitis, constitutive and TNF-alpha-induced expression of MAdCAM-1 in the intestine, cecum, and colon was not different from those values obtained with healthy wild-type controls. IL-10-deficient mice with active colitis exhibited a four- to fivefold greater expression of MAdCAM-1 in the cecum and colon compared with their healthy controls or to IL-10 k/o mice with no evidence of colitis. Taken together, these data demonstrate that TNF-alpha enhances surface expression of MAdCAM-1 in intestinal and colonic tissues to the same extent in both wild-type and IL-10 k/o mice with no colonic inflammation, whereas IL-10 k/o mice with active colitis exhibited a profound up-regulation of MAdCAM-1 in the colon.

Role of tumor necrosis factor and interferon gamma in endotoxin-induced E-selectin expression.

Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), potent inflammatory cytokines, are released by macrophages during endotoxin shock. However, the contribution of these cytokines to endotoxin-induced inflammation has not been defined. The expression of E-selectin, measured using the dual radiolabeled monoclonal antibody (mAb) technique, was monitored in different tissues of endotoxin-challenged wild-type and IFN-gamma-deficient mice receiving a mAb to TNF-alpha (TN3). A significant elevation in E-selectin expression occurred in all tissues of wild-type mice challenged with endotoxin. Injection of TN3 in wild-type mice significantly attenuated the endotoxin-induced up-regulation of E-selectin in all tissues (p < .05) except the pancreas. The level of reduction in endotoxin-induced E-selectin expression ranged between 30% in the stomach to 60% in the small intestine. E-selectin expression in endotoxin-challenged, IFN-gamma-deficient mice was significantly reduced in the small and large intestines, when compared with endotoxin-challenged wild-type mice. Although IFN-gamma deficiency had no effect on E-selectin expression in other tissues, administration of TN3 to endotoxin-challenged, IFN-gamma-deficient mice significantly reduced E-selectin expression to levels observed in endotoxin-challenged, wild-type mice that received TN3. These findings indicate that TNF-alpha is essential for achievement of maximal E-selectin expression in most vascular beds during endotoxemia, whereas the contribution of IFN-gamma is largely confined to the small intestine.

Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen.

Expression of E-selectin and P-selectin is critical in the effector phase of leukocyte recruitment in response to Ag. Whether their relative roles differ between tissues in response to the same Ag is unknown. In this study, a type I hypersensitivity response was elicited in C57BL/6 mice by systemic sensitization with OVA. Following local Ag challenge, endothelial selectin expression was examined in the skin and cremaster muscle microvasculature using a dual-radiolabeled mAb technique. Next, the dermal and muscle microcirculations were visualized using intravital microscopy to establish roles for P-selectin and/or E-selectin. In untreated mice, leukocyte recruitment in both skin and skeletal muscle was mediated entirely by P-selectin. Following Ag challenge, leukocyte rolling flux and adhesion were dramatically increased and leukocyte rolling velocity was unchanged in muscle. Only P-selectin expression increased in muscle, and leukocyte recruitment was entirely dependent upon this selectin. In contrast, in Ag-challenged skin, leukocyte rolling flux did not increase, but rolling velocity dropped profoundly. In skin, only E-selectin expression increased, and blockade of either E-selectin or P-selectin had minimal effect on either rolling flux or rolling velocity. Blockade of both selectins reduced rolling flux by 80% and increased rolling velocity sevenfold. These data highlight striking differences in expression of the endothelial selectins in separate microvascular beds in response to the same stimulus and demonstrate that these differences underlie very different patterns of leukocyte recruitment. The data underscore the importance of studying individual microvascular beds to understand tissue-specific leukocyte recruitment in vivo.

In vivo measurements of endothelial cell adhesion molecule expression.

The essential role of endothelial CAMs in the recruitment of leukocytes to sites of inflammation has been extensively studied in recent years. The development of techniques such as the radiolabeled MAb method that is described in this chapter has enabled investigators to quantitatively assess the expression of endothelial CAMs in different regional vascular beds. Although the study of adhesion molecules in vitro has extended our knowledge of the mechanisms and mediators of endothelial CAM function, the newly acquired in vivo data would indicate that findings resulting from experiments on monolayers of cultured endothelial cells may not accurately depict inflammatory events that occur in vivo. Thus, additional work is needed to more fully define the conditions that account for the discrepencies noted between the in vivo and in vitro models of endothelial CAM expression.

Differential expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) in murine tissues.

OBJECTIVE: To characterize and compare the expression of PECAM-1 in unstimulated and tumor necrosis factor alpha-(TNF-alpha)-stimulated tissues of mice. METHODS: Binding and non-binding monoclonal antibodies (mAb) were radiolabeled and injected into wild-type mice and mice deficient of P-selectin, CD18, or ICAM-1. The relative accumulation of binding mAb (PECAM-1 mAb) was determined in wild-type mice following a 25 micrograms/kg i.p. injection of TNF-alpha and in mutant mice under basal conditions. RESULTS: Under unstimulated conditions, PECAM-1 was significantly expressed in all tissues examined, with no changes occurring after TNF-alpha stimulation. An equivalence of PECAM-1 expression was observed in unstimulated tissues among wild-type mice and mice that are genetically deficient in either CD18, ICAM-1, or P-selectin. The level of PECAM-1 expression in different vascular beds was highly correlated to published values of endothelial surface area. Normalization of previously published values of ICAM-1, VCAM-1, E- and P-selectin expression relative to PECAM-1 expression in the same tissues revealed a diminished organ-to-organ variability in expression of the different adhesion molecules. Estimates of adhesion molecule expression in lung and brain were most profoundly affected by normalization to PECAM-1 expression. CONCLUSIONS: These findings support the use of PECAM-1 expression in regional vascular beds as an indicator of endothelial cell surface area.

Interleukin-12-induced adhesion molecule expression in murine liver.

Systemically administered interleukin (IL)-12 causes liver inflammation in mice characterized by Kupffer cell proliferation and hypertrophy, hepatocyte necrosis, and multifocal accumulations of leukocytes in the hepatic parenchyma and around portal tracts and central veins. We have used both immunohistochemical staining and radiolabeled antibody quantitation to examine adhesion molecule expression in the livers of mice dosed daily with murine IL-12. Cells infiltrating livers of IL-12-treated mice were primarily mononuclear leukocytes expressing LFA-1, VLA-4, MAC-1, and CD18 adhesion molecules but little L-selectin. Kupffer cells constitutively expressed LFA-1 and smaller amounts of MAC-1, and high levels of ICAM-1 were constitutively expressed by liver sinusoidal lining cells, portal tract, and central vein endothelia. With IL-12 treatment, existing ICAM-1 expression was up-regulated and de novo expression occurred along bile duct epithelia. VCAM-1 levels were dramatically increased, with induced expression occurring along portal tract and central vein endothelia and scattered bile duct epithelial cells and in aggregations of cells in perivascular areas and the liver parenchyma. Although constitutive expression of E- and P-selectin was negligible, Il-12 induced a moderate rise in E-selectin levels. These increases in adhesion molecule expression may have implications for the therapeutic use of IL-12, especially in patients with liver disease or autoimmune conditions where augmented adhesion molecule expression may be critical to disease pathogenesis.

Endothelial cell adhesion molecule expression in gene-targeted mice.

Gene-targeted mice are now routinely employed as tools for defining the contribution of different leukocyte and endothelial cell adhesion molecules to the leukocyte recruitment and tissue injury associated with acute and chronic inflammation. The objective of this study was to determine whether gene-targeted mice that are deficient in CD11/CD18, intracellular adhesion molecule-1 (ICAM-1), or P-selectin exhibit an altered constitutive or induced expression of the endothelial cell adhesion molecules E- and P-selectin. The gene-targeted mice were all developed in the 129Sv mouse strain and backcrossed into C57B1/6J mice. The number of backcrosses ranged between 8 (P-selectin) and 10 (CD18 and ICAM-1) generations. The dual-radiolabeled monoclonal antibody technique was used to quantify E- and P-selectin expression in different vascular beds. In the unstimulated state, E-selectin expression was significantly elevated (relative to wild-type mice) in the stomach, large intestine, and brain of mutants deficient in ICAM-1. In general, constitutive expression of P-selectin did not differ between wild-type, ICAM-1-deficient, and CD11/CD18-deficient mutants. In CD11/CD18-deficient mice, tumor necrosis factor-alpha (TNF-alpha) administration elicited a more profound upregulation of P-selectin in several vascular beds, compared with wild-type and ICAM-1-deficient mice. E-selectin expression in brain of TNF-alpha-stimulated, ICAM-1-deficient, and P-selectin-deficient mice was attenuated compared with wild-type mice. These findings indicate that chronic deficiency of some of the adhesion glycoproteins that mediate leukocyte recruitment alters basal and induced surface expression of other adhesion molecules on endothelial cells.

Lung neutrophil retention and injury after intestinal ischemia/reperfusion.

OBJECTIVE: To define the mechanisms responsible for the lung leukosequestration and injury elicited by intestinal ischemia/reperfusion (I/R). METHODS: The effect of 120 minutes of superior mesenteric artery occlusion and 90 minutes of reperfusion on neutrophil deformability, lung neutrophil retention, and pulmonary microvascular permeability was determined. RESULTS: Compared with control surgery, intestinal I/R resulted in a significant increase in neutrophil stiffness (mean yield pressure [Pyield], 1.533 +/- 0.075 and 2.302 +/- 0.288 cm H2O, respectively) and lung neutrophil content (6.3 +/- 1.4 and 31.5 +/- 6.4 U/g wet weight, respectively). These changes were not affected by inhibition of neutrophil adherence before gut reperfusion. However, the increased lung microvascular permeability elicited by gut I/R (0.111 +/- 0.020 [control surgery] and 0.255 +/- 0.041 [I/R] mL/min/cm H2O/100 g lung tissue) was significantly attenuated by administration of antibodies directed against neutrophil or endothelial determinants of leukocyte adhesion. CONCLUSIONS: The results of this study suggest that intestinal I/R is a potent inflammatory stimulus that elicits an increase in neutrophil stiffness and lung neutrophil retention independent of neutrophil-endothelial cell adhesion. In contrast, the increased lung microvascular permeability elicited by gut I/R is attenuated by strategies that interfere with neutrophil-endothelial cell adhesion.

Ischemia/reperfusion-induced leukocyte-endothelial interactions in postcapillary venules.

The recruitment of leukocytes into postcapillary venules following reperfusion of ischemic tissues is a classical inflammatory response that is influenced by a balance of adhesive and hemodynamic forces. Initial periods of reperfusion are characterized by an elevation in low affinity adhesive interactions between leukocytes and endothelial cells, which permit the leukocytes to establish intimate contact with the vascular endothelial lining as they roll across it. This rolling behavior of leukocytes increases the probability that stronger adhesive interactions between leukocyte and endothelial cell will occur, allowing the leukocyte to firmly adhere to the endothelium and subsequently to emigrate across the venular wall into the interstitial compartment. Several factors appear to contribute to the leukocyte-endothelial cell adhesion that is elicited by ischemia/reperfusion, including: 1) the magnitude of adhesion molecule expression on leukocyte and/or endothelial cell surfaces, 2) reactive oxygen metabolites and lipid mediators released from stimulated leukocytes and endothelial cells and 3) hemodynamic dispersal forces that act to sweep leukocytes from the microvessel wall. Intravital microscopic techniques have allowed investigators to focus on inflammatory responses in postcapillary venules, which is the primary site of leukocyte adhesion and vascular protein leakage. This technology has also been employed to delineate the role of adhesive and hemodynamic factors in promoting leukocyte rolling, adhesion, and emigration in postcapillary venules exposed to ischemia/reperfusion.

Complement-mediated lung injury and neutrophil retention after intestinal ischemia-reperfusion.

Complement-mediated neutrophil activation appears to play an important role in ischemia-reperfusion (I/R) injury in a variety of tissues, including the heart, lung, and small bowel. The objective of this study was to determine whether inhibition of the alternative and classic complement cascades by administration of soluble complement receptor 1 (sCR1) prevents the increased neutrophil stiffness, lung neutrophil retention, and pulmonary microvascular injury elicited by a systemic inflammatory insult. Isolated lungs were perfused with blood obtained from animals subjected to 2 h of intestinal ischemia and 20 min of reperfusion (I/R) or control (nonischemic) surgery. Intestinal I/R resulted in a significant increase in neutrophil stiffness, lung neutrophil retention, and increased pulmonary microvascular permeability, effects that were prevented by administration of sCR1 before intestinal reperfusion. The results of this study suggest that I/R injury in the gut is a potent systemic inflammatory stimulus that induces complement-mediated neutrophil stiffness, lung neutrophil entrapment, and pulmonary microvascular dysfunction.

Modulation of P-selectin expression in the postischemic intestinal microvasculature.

The dual radiolabeled monoclonal antibody technique was used to 1) define the magnitude and kinetics of P-selectin expression in murine small intestine exposed to ischemia-reperfusion (I/R), and 2) determine the factor(s) responsible for initiating this response. Within 10 min after release of a 20-min arterial occlusion, intestinal P-selectin expression increased two- to threefold compared with control values. Peak (4-fold) expression of P-selectin was noted at 5 h after reperfusion, returning to the control value at 24 h. The early (10-30 min) I/R-induced upregulation of P-selectin appears to reflect mobilization of a performed pool of the adhesion molecule, whereas the later (5 h) rise appears to be transcription dependent. The early increase in P-selectin expression was not inhibited by pretreatment with either oxypurinol (inhibits xanthine oxidase), diphenhydramine (H1-receptor antagonist), or MK-571 (leukotriene C4/D4 antagonist), nor was it blunted in transgenic mice expressing three times the normal level of copper-zinc superoxide dismutase or in mast cell-deficient mice. However, significant inhibition was noted after treatment with either MK-886 (5-lipoxygenase inhibitor) or a nitric oxide (NO) donor (diethylenetriamine/NO). These findings indicate that the early I/R-induced increase in intestinal P-selectin expression is mediated by a 5-lipoxygenase-dependent NO-inhibitable mechanism.

Heterogeneity of expression of E- and P-selectins in vivo.

A novel technique involving radiolabeled monoclonal antibodies was used to characterize and compare the expression of E- and P-selectin on unstimulated, histamine-challenged, and endotoxin-challenged endothelial cells in various tissues of the mouse. Under unstimulated conditions, E-selectin was absent in all organs, but significant expression of P-selectin was observed in several organs. Histamine induced a rapid time-dependent upregulation of P-selectin, with the largest responses observed in mesentery and lung. Significant fold elevations in P-selectin expression occurred as early as 5 minutes after the histamine injection and remained elevated up to 1 hour. Histamine-induced P-selectin upregulation was inhibited by the H1 receptor antagonist diphenhydramine, whereas the H2 receptor antagonist cimetidine had no effect. Endotoxin (lipopolysaccharide [LPS]) also induced a time-dependent expression of P-selectin that reached a maximum between 4 and 8 hours after endotoxin administration. LPS-induced upregulation of P-selectin was greatest in heart and stomach, which exhibited insignificant constitutive expression of P-selectin. LPS also induced a time-dependent upregulation of E-selectin, with maximal expression occurring 3 to 5 hours after intraperitoneal administration. The lung and small intestine exhibited the largest responses to LPS challenge. Histamine administration did not affect E-selectin expression in any tissue. E- and P-selectin-deficient mice were used to test the specificity of monoclonal antibody binding in unstimulated, histamine-challenged, and LPS-stimulated tissues. Vascular binding of the radiolabeled E-selectin and P-selectin monoclonal antibodies was not observed in the respective deficient mice. These findings suggest that P-selectin is constitutively expressed on vascular endothelium in some tissues of the mouse and that there are significant regional differences in the magnitude and time course of histamine- and endotoxin-induced P-selectin expression. In contrast, E-selectin appears to be absent on unstimulated vascular endothelium but is upregulated within 3 hours after the administration of endotoxin in most tissues.

Effects of leukocyte-capillary plugging on the resistance to flow in the microvasculature of cremaster muscle for normal and activated leukocytes.

The effects of leukocyte (WBC) sequestration in the capillary network on resistance to flow (RA-V) were obtained during bolus infusions of WBCs in cremaster muscle (hamster). RA-V was calculated from simultaneous measurements of arteriole to venule pressure drop and arteriolar red cell velocity. Bolus infusions of red cells (RBCs) alone resulted in a 5% decrease in RA-V, due to the clearance of circulating WBCs from the network. Infusions of RBCs with leukocrits of one to nine times systemic resulted in insignificant transient increase in RA-V of 5 to 10%. The effect of WBC activation was studied by their incubation in N-formyl-methionyl-leucyl-phenylalanine (FMLP) to activate (and stiffen) the polymorphonuclear WBCs (PMNs) or phorbal myristate acetate (PMA) to activate all WBCs in the bolus. Compared with normal WBCs, infusions of mixtures of RBCs and activated WBCs had no significant effect on the transient increase in resistance as the bolus traversed the capillary network. However, mixtures with either normal or FMLP-treated WBCs increased the steady state RA-V in proportion to the cumulative number of WBCs infused, due to residual capillary plugging following washout of the bolus. The cumulative infusion of 20 x 10(6) normal or FMLP-activated WBCs resulted in a 25% increase in RA-V above baseline. With PMA activation, cumulative infusions of only 5 x 10(6) WBCs in the RBC suspension also resulted in a 25% increase in RA-V, which was three times the increase obtained for an equal number of FMLP-activated WBCs. Following the cumulative infusion of 12 x 10(6) PMA-activated WBCs, RA-V increased inordinately to approximately 250% of baseline. These substantially greater increase in capillary plugging and RA-V with PMA activation were in accord with the threefold greater number of stiffened lymphocytes (which do not respond to FMLP) relative to PMNs in the boli. Thus, capillary plugging by activated WBCs may have a far greater detrimental effect on blood flow through the microvasculature compared to normal WBCs, and the extent of this effect is strongly dependent on the number of activated WBCs in the circulation.

The mean filtration pressure of leukocyte suspensions and its relation to the passage of leukocytes through nuclepore filters and capillary networks.

OBJECTIVE: To quantitatively evaluate the deformability of bulk suspensions of leukocytes (WBCs), account for the variance of individual cell mechanical properties, and deduce the potential for WBC entrapment within the capillary network in response to alterations in cell properties. METHODS: The transient washout of WBCs initially trapped under low perfusion pressure in 5-microns pores of Nuclepore filters was analyzed for a 0.2-ml bolus of WBCs (derived from hamsters) with equal numbers of filter pores and cells, to characterize the statistical distribution of pressures, Pyield, required to dislodge the cells. Contributions of the variance in WBC diameter, pore diameter, and cremaster muscle capillary diameter to Pyield were estimated with a WBC cortical shell model and an analysis of the probability function of the ratio of WBC to pore diameter, lambda. RESULTS: For normal WBCs, Pyield exhibited a log-normal distribution with mean (Pyield) of 0.59 cmH2O. Incubation of cells in cytochalasin-B reduced (Pyield) almost 50%, whereas phorbol myristate acetate increased (Pyield) twofold. Incubation in N-formyl-methiolnyl-leucyl-phenylalanine had no significant effect on (Pyield), as polymorphonuclear cells became permanently trapped in the filter. The fluorescent dyes acridine orange, acridine red, and tetramethylrhodamine isothiocyanate increased (Pyield) as much as 10-fold, whereas steady-flow filtration methods showed no alteration. Analysis of the distribution of lambda revealed that due to their smaller pore diameters, in vitro filtration methods may overestimate in vivo values of (Pyield) by almost twofold. CONCLUSIONS: The transient filtration of WBC suspensions appears to be much more sensitive to subtle alterations in WBC deformability than steady-flow methods and may provide greater insight into the determinants of capillary perfusion. Estimates of (Pyield) are comparable to those obtained with micropipettes and permit analysis of substantially greater numbers of cells within a sample. Fluorescence labeling techniques should be used with caution, as they may dramatically alter cell properties to an extent undetectable by direct in vivo observations.

Leukocyte sequestration in the microvasculature in normal and low flow states.

Techniques of indicator dilution were applied to determine the relative transit times (TTs) of fluorescently labeled leukocytes (TTWBC), red blood cells (TTRBC), and plasma (TTpl) between functionally paired arterioles and venules in hamster cremaster muscle in normal and low flow states. In the normal flow state, paired measurements of TTWBC/TTpl in arteriovenous (A-V) pairs bounding the true capillaries averaged 0.75 +/- 0.08 (SE) s and were not significantly different from an average TTRBC/TTpl of 0.78 +/- 0.06 (SE) s as WBCs appeared to traverse the capillary segment through more centralized preferential pathways. In larger-diameter A-V pairs, significantly larger (10%) values of TTWBC/TTpl were found compared with TTRBC/TTpl due to margination of WBCs in postcapillary venules. To assess the relative effects of WBC-capillary plugging and WBC adhesion in venules on flow resistance, TTWBC was measured in normal and low flow states, with the latter induced by systemic administration of sodium nitroprusside, which resulted in an increase in TTpl from a norm of 1.08 +/- 0.16 to 2.62 +/- 0.44 (SE) s (P < 0.05). With onset of the low flow state, TTWBC/TTpl, the number of plugged capillaries, and the duration of capillary plugs did not change significantly from the norm. In contrast, the rate at which WBC-endothelium (-EC) adhesion increased with successive bolus injections increased approximately eight-fold during hypoperfusion. Estimates of the percentage increase in segmental resistance at the capillary level (due to plugging) and in postcapillary venules (due to EC adhesion) revealed that venous resistance may increase at a threefold greater rate due to WBC sequestration with each successive bolus infusion. Inasmuch as hemodynamic resistance in capillary and venular segments is of the same order of magnitude in the normal flow state, it appears that WBC adhesion in venules may have a far greater deleterious effect on microvascular blood flow in the low flow state.

Effect of erythrocyte deformability on in vivo red cell transit time and hematocrit and their correlation with in vitro filterability.

Indicator dilution techniques were applied to measure mean transit time of fluorescently labeled red blood cells (RBCs) (TTRBC) and plasma (TTpl) between functionally paired arterioles and venules (A-V) in cremaster muscle (rat) for normal RBCs and cells hardened by in vitro incubation in graded concentrations of glutaraldehyde. Dispersion of a bolus introduced into the contralateral femoral artery permitted computation of TT by cross-correlation of fluorescence intensity-time curves in A-V pairs. Parallel in vitro assessments of RBC deformability were made by filtration through 5-microns pore Nuclepore filters to express deformability in terms of the ratio of resistance to flow through a pore with RBCs present to that with suspending medium alone, beta. The average microvascular hematocrit (Hmicro) normalized with respect to systemic hematocrit (Hsys) was calculated from TTRBC and TTpl. For 26 A-V pairs of the third and fourth orders of branching, TTRBC averaged 0.63 sec for normal control cells (beta = 2.61), and TTpl averaged 0.85 sec with an average TTRBC/TTpl equal to 0.85. The corresponding value of Hmicro/Hsys was significantly < 1 and averaged 0.87. This greater value of Hmicro/Hsys compared to direct measurements in the literature was attributed to the unique ability of the indicator dilution technique to account for red cell flux throughout the network. For hardened RBCs with beta < 10, TTRBC/TTpl and Hmicro/Hsys increased on average 30%, but were weakly correlated with increasing beta due to redistribution of RBCs throughout pathways of lesser resistance. However, as beta rose from 10 to 20, these pathways became overwhelmed by hardened RBCs and TTRBC/TTpl increased threefold due to retardation of the RBC flux, with a concomitant rise in Hmicro/Hsys. These results clearly demonstrate the extent to which diminished RBC deformability of a magnitude found in clinical disorders may affect microvascular perfusion.