Effect of zymosan-activated of rabbit polymorphonuclear HIDETAKA
plasma on the deformability leukocytes
University of British Columbia Pulmonary Research Laboratory, St. Paul’s Hospital, Vancouver, British Columbia V6Z 1 Y6, Canada; and Herman B. Wells Center for Pediatric Research, Section of Pulmonology and Intensive Care, Department of Pediatrics, Riley Hospital for Children, Indiana University, Indianapolis, Indiana 46202-5225 INANO,HIDETAKA,DEANENGLISH,ANDCLAIRE M. DOERSdiated through a decrease in the deformability of the CHUK. Effect of zymosan-activated plasma on the deformability PMN. Inflammatory stimuli that activate PMN can of rabbit polymorphonuclear leukocytes. J. Appl. Physiol. 73(4): cause a rapid decrease in the deformability of the PMN 1370-1376,1992.-Intravascular infusion of inflammatory me(9, 12, 16, 22, 34). This decreased deformability is diators causes a sudden neutropenia due to the sequestration of thought to be mediated by a rapid polymerization of solupolymorphonuclear leukocytes (PMN) within the microvascuble G-actin to filamentous F-actin at the cell periphery, lature of the lung and other organs. This sequestration could be increasing the rigidity and the viscosity of the PMN (9, due to a decrease in the ability of PMN to deform and pass 12,24,25,27,33,34). Decreased deformability is likely to through the narrow capillary bed. The purpose of this study alter the ability of the PMN to travel through the pulmowas to determine if the complement fragments present in zynary capillaries, many of which are narrower than the mosan-activated plasma (ZAP) caused a rapid stiffening of PMN. The PMN deformability was determined by measuring spherical diameter of the PMN (17). Inability to pass the pressure required to pass PMN through a polycarbonate through the pulmonary capillary bed would result in a filter containing 5-pm pores at a constant flow rate as well as sudden sequestration of PMN and neutropenia. the extraction of PMN compared with red blood cells and 1251The purpose of this study was to determine whether labeled albumin by the filter. The role of the cytoskeleton in the complement protein 5 fragments present in zymoPMN deformation was examined in studies where F-actin forSan-activated plasma (ZAP) cause a decrease in the abilmation was inhibited using cytochalasin B or microtubule asity of PMN to deform, as measured by the pressure resembly was inhibited using colchicine. The results showed that quired to pass the PMN through a polycarbonate filter at treatment with ZAP induced a rapid decrease in PMN deforma constant flow rate (9,10,12,16,22,23,29,34). The role ability. Inhibiting the formation of F-actin made the unstimulated PMN more deformable and reduced the stiffening inof the cytoskeleton in this change was examined in studduced by ZAP. In contrast, inhibition of microtubule reassemies where F-actin formation was inhibited using cytochably did not alter either normal deformability or the lasin B (12,33,34) or microtubule assembly was inhibited ZAP-induced decrease in deformability. In vivo, colchicine inusing colchicine (1, 2, 4, 19, 21, 32, 35). These studies creased normal PMN margination but did not inhibit the rapid show that inhibiting the formation of F-actin made the sequestration of PMN induced by infusion of ZAP. These studunstimulated PMN more deformable and reduced the ies indicate that ZAP induces a rapid decrease in PMN deformstiffening induced by ZAP. In contrast, inhibition of miability that is mediated through the cytoskeleton. They suggest crotubule reassembly did not alter either the normal dethat this decrease is due to the polymerization of F-actin. cytoskeleton;
5a; actin; microtubules
PMN)playarole in the pathogenesis of acute lung injuries, such as the adult respiratory distress syndrome, by sequestering in the lung and injuring the alveolocapillary membrane (3, POLYMORPHONUCLEARLEUKOCYTES(
This injury can cause an increase in vascu-
lar permeability, resulting in pulmonary edema. Intravascular infusion of inflammatory mediators, including active complement protein 5 fragments and formyl-methionyl-leucyl-phenylalanine, causes a rapid sequestration of PMN (6, 13, 15, 18, 31, 34). The mechanism through which this sequestration occurs could involve an increase in the adhesion of PMN to endothelial ceils. However, we and other laboratories have shown that this sequestration is not dependent on CDll/CD18-mediated adhesion (8, 20). Alternatively, PMN sequestration may be me1370
0161-7567/92 $2.00 Copyright
formability or the ZAP-induced decrease in deformability. These studies indicate that ZAP induces a rapid decrease in PMN deformability that is ‘mediated through the cytoskeleton. They suggest that this decrease is due to the polymerization of F-actin. METHODS
Rabbit PMN were isolated from peripheral blood using previously described methods (6,7). In brief, dextran (l2 X lo5 mol wt, 1.7% final concentration) was combined with blood anticoagulated with acid-citrate-dextrose to sediment the red blood cells (RBC). After hypotonic lysis in water for 20 s, the PMN were separated from the mononuclear cells by centrifugation through Histopaque (Sigma 1007, St. Louis, MO). The PMN purity was >95%.
1992 the American
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ZAP was prepared by incubating heparinized rabbit plasma combined with zymosan A yeast (5 mg/ml plasma) at 37°C for 30 min (6). The plasma was centrifuged twice at 500 g for 10 min.
number of cells or radioisotope counts injected. The area under the curves to the peak of the reference (RBC or albumin) curve was determined. The extraction was calculated as PMN extraction
Filtration Assay Two types of assays were used to determine the deformability of PMN, both of which measured the pressure needed to pass PMN through a polycarbonate filter with a uniform pore diameter of 5 pm (Nucleopore Canada). In the first, a suspension of PMN was continuously passed through the filter. In the second, the PMN were mixed with RBC and radiolabeled albumin and injected , as a bolus. Continuous infusion assay. Three groups were studied: group 1, PMN + 5% normal plasma (control); group 2, PMN + 5% ZAP; and group 3, glutaraldehyde-fixed PMN + 5% normal plasma. After rabbit PMN were mixed with either 5% normal plasma (group 1) or ZAP (group 2) or 1.25% glutaraldehyde-fixed PMN with 5% normal plasma (group 3) at a concentration of 4.1 t 0.9 X lo5 PMN/ml for 2 min, they were passed through a polycarbonate filter at a constant flow rate of 4 ml/min for 2.5 min. The filtrate was collected at 7.5-s intervals. The pressure across the filter was measured using a differential pressure transducer. The PMN concentration in the input and in each fraction of the filtrate was measured using a Coulter counter. The filter was fixed using 2.5% glutaraldehyde. Onehalf the filter was stained with toluidine blue 0, placed on a microscope slide, and examined en face. The other half was embedded in glycol methacrylate, and cross sections were cut. The number of PMN retained in the filter was calculated by counting the number of PMN that was present in five random fields for each filter. These fields were digitized to determine the area (Bioquant BQ System IV, R & M Biometrics). The total number of PMN in the filter was calculated by multiplying the number of PMN per square micrometer of filter times the total area of the filter that was accessible to the flow of PMN. The cross-sectioned filter was examined to determine the fraction of the PMN that was on the surface of the filter (%outside) or within the pores of the filter (%inside). The location of the PMN that were inside the filter was categorized as within the proximal (entering), middle, or distal (exiting) one-third of the pore length. Bolus injection assay. Two groups were studied: group 1, PMN + 5% normal plasma, and group 2, PMN + 5% ZAP. Isolated rabbit PMN (0.2 ml of 4.1 t 0.5 X 106/ml) were mixed with a similar number of RBC and 0.1 &i 1251-albumin. After incubating in ZAP or normal plasma for 2 min, the mixture was injected as a 0.2-ml bolus into a port proximal to the filter. This bolus was immediately followed by a continuous infusion of phosphate buffer at 2 ml/min for 1.5 min. The differential pressure across the filter was measured, and the filtrate was collected at 3.5-s intervals. The extraction by the filter of PMN compared with albumin and with RBC was calculated by constructing time vs. concentration curves normalized for the total
area under PMN curve area under reference curve
The number of PMN retained in the filter was quantitated histologically using the method described above. Deformability
of PMN in Whole Blood
The effect of ZAP on PMN deformability in whole blood was measured using the bolus injection assay. Rabbit blood was drawn into heparin (30 U/ml blood). Either ZAP or plasma was added to blood (final concentration of 5%) for 2 min. PMN deformability was measured using the bolus injection assay. Inhibition of F-Actin Formation and Microtubule Assembly Isolated rabbit PMN were divided into eight aliquots of 4 X 106/ml. The effect of cytochalasin B and colchicine on PMN deformability was examined in the following eight groups (n = 5 in each group): group 1, PMN + 5% normal plasma (control); group 2, PMN + 5% ZAP; group 3, PMN + 50 ,uM colchicine + 5% normal plasma; group 4, PMN + 50 PM colchicine + 5% ZAP; group 5, PMN + 0.2% dimethyl sulfoxide (DMSO) + 5% normal plasma (control); group 6, PMN + 0.2% DMSO + 5% ZAP; group 7, PMN + 10 PM cytochalasin B + 5% normal plasma; and group 8, PMN + 10 PM cytochalasin B + 5% ZAP. Isolated PMN were incubated with colchicine (Sigma; final concentration 50 PM in saline) for 30 min at room temperature before treatment with plasma or ZAP. Cytochalasin B (Sigma) was dissolved in DMSO and mixed with the isolated PMN for 5 min at room temperature (final concentration 10 PM cytochalasin B and 0.2% DMSO). The bolus injection assay was started after 2 min of treatment with plasma or ZAP. The bolus injection assay was performed to measure the differential pressure required to pass PMN (0.2 ml of 4 X 106/ml) through a polycarbonate filter. The bolus of the mixture was immediately followed by a continuous infusion of phosphate buffer at 2 ml/min, and the peak pressure was compared among the groups. Effect of Inhibition of Microtubule Assembly on ZAP-induced Neutropenia New Zealand White rabbits weighing 3.3 t 0.4 (SD) kg were anesthetized with ketamine hydrochloride (80-100 mg/kg im) and acepromazine maleate (8-10 mg/kg im). Catheters were inserted in the left carotid artery, the right jugular vein, and the marginal ear vein. The animals received a tracheostomy and breathed room air. Heparin (200 U/kg) was injected into the left carotid artery. Three groups of animals were studied: group 1, animals pretreated wi th colchicine (1 mg /kg in saline) that received infusions of normal pllasma (n = 5); group 2, animals pretreated with colchicine (1 mg/kg in saline) that received infusions of ZAP (n = 5); and group 3, ani-
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loo2 I: k
5 60 ko ii 40 z E 20
1 1 .o Time
1 .o Time
FIG. 1. Differential pressure across a 5-pm polycarbonate filter during passage of polymorphonuclear leukocytes (PMN). Pressure gradient was increased when PMN were treated with zymosan-activated plasma (ZAP) (A) compared with normal plasma (0), although shape of pressure curve was similar. In contrast, increase was slower and no plateau was reached in filtrate pressure of glutaraldehyde-fixed group (O), resulting in very large pressure gradients at end point. Data are means + SE. *P < 0.05 between plasma(group I) and ZAP-treated (group 2) PMN. #P < 0.05 between plasma-treated (group I) and fixed (group 3) PMN.
mals pretreated with saline (1 ml/kg) that received infusions of ZAP (n = 5). After baseline (time -10) samples of blood was drawn, colchicine (group 1 and 2) or saline (group 3) was injected into the right jugular vein. Blood samples for cell counts were taken after 2 (time -8), 4 (time -6), 7 (time -3), and IO (time 0) min. Intravenous infusions of ZAP (group 2 and 3) or normal plasma (group 1) were begun through the ear vein at a rate of 1 ml/min for 15 min (from time 0 to 15 min). Blood samples were taken 0.5,1,2,4,7,11,15, 20, 25, 30, 45, and 60 min after plasma or ZAP infusion. Blood volume was replaced by saline at each time point. Circulating blood cell counts were corrected for hematocrit and expressed as percent change in corrected values from the baseline (time -10) values prior to colchicine. Statistics
A repeated analysis of variance was used to compare the differential pressure, the recovery of PMN in the filtrate, and the leukocyte counts over time between the groups with corrections for multiple comparisons where necessary. Paired t tests were used to compare the number of PMN in the filter and the peak pressure of PMN or whole blood. Paired t tests corrected for multiple comparisons were used to compare the extraction of PMN with respect to RBC and 1251-albumin and to compare the peak pressure in the studies examining the effect of colchicine or cytochalasin B. A probability