Leukocyte and platelet margination within microvasculature of rabbit lungs C. M. DOERSCHUK, G. P. DOWNEY, D. E. DOHERTY, M. OHGAMI, G. S. WORTHEN, P. M. HENSON, AND
D. ENGLISH, J. C. HOGG
R. P. GIE,
The University of British Columbia Pulmonary Research Laboratory, Vancouver, British Columbia V6Z 1 Y6, Canada; and Departments of Medicine and Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Service of Medicine, Denver Veterans Administration Hospital, and Departments of Pathology and Medicine, Section of Pulmonary Diseases, University of Colorado School of Medicine, Denver, Colorado 80206
DOERSCHUK, C. M., G. P. DOWNEY, D. E. DOHERTY,D. ENGLISH, R. P. GIE, M. OHGAMI, G. S. WORTHEN,P. M. HENSON, AND J. C. HOGG. Leukocyte and platelet margination within microvasculuture of rabbit lungs. J. Appl. Physiol. 68(5): 1956-1961, 1990.-These studiescompare the behavior of radiolabeled neutrophils, monocytes, lymphocytes, and platelets during their first passthrough the pulmonary circulation after a central venous injection and their distribution within the circulation 10min later. Their first passthrough the pulmonary circulation was comparedwith erythrocytes (RBCs) using the indicator-dilution technique, and their recovery within the circulation of the lung and other organswas determined at 10 min by counting the radioisotopesin eachorgan. The extraction of each cell relative to RBCs during the first passthrough the lung correlated with cell size in that the neutrophils (volume 107-140fl) showed97.6 t 0.6% extraction, monocytes(volume 80-105 fl) showed91.4t 1.7%extraction, lymphocytes (volume 36-75 fl) showed80.1 2 4.4% extraction, and platelets (volume 4-7 fl) showed33.1 t 3.9% extraction. After 10 min of circulation, the proportion of injected cells remaining in the lung was similar for neutrophils and monocytes (27.4 k 1.8 vs. 31.4 t 1.6%) but lower for lymphocytes (18.6 k 2.9%) and platelets (3.1 t 0.5%). All of the leukocytes were found to have a substantial marginatedpool within the lung, whereasthe platelets did not. The exchangebetweenthe circulating and marginated pools of leukocytes in the lung was related to blood velocity, with the least retention occurring in lung regionswith shortest RBC transit times. We conclude that cell size is a major factor determining the time that cells will be delayed by the pulmonary microvasculature. However, other factors such as the cells’ deformability and their interaction with the endothelium may alsobe important.
neutrophils; monocytes; lymphocytes; platelets; erythrocyte transit time; pulmonary marginated leukocyte pool
THE PULMONARY capillary
bed is made up of a large number of short interconnected segments that range in size from 2 to 12 m in diameter so that some are narrow enough to restrict the passage of the circulating cells (1, ll,l3). Although erythrocytes and leukocytes have similar diameters, the erythrocytes can move through these restrictions much more easily because they are more deformable (3,11,13,16,22,23,25). The multisegmented nature of the pulmonary capillary bed allows the eryth1956
0161-7567/90 $1.50 Copyright
0
rocytes to stream around the neutrophils, which are delayed in these restrictive segments. This concentrates the neutrophils with respect to erythrocytes, which accounts for the large marginated pool of neutrophils in the pulmonary capillary bed (5, 11, 13). The purpose of this study was to extend our previous observations on neutrophils to lymphocytes, monocytes, and platelets to determine if these cells also marginate within the lung microvessels. METHODS Isolation and labeling of leukocytes. The methods for isolating neutrophils (PMNs), monocytes, lymphocytes, and platelets used in this study have been fully described elsewhere (6, 9, 28, 30). Briefly, titrated whole blood, collected by sterile puncture of the central ear artery of unanesthetized New Zealand White rabbits, was centrifuged at 300 g for 20 min. The platelet-rich plasma was aspirated and centrifuged at 2,500 g for 20 min to yield platelet-poor plasma (PPP). The remaining cells were sedimented with dextran (molecular wt 500,000, Pharmacia Fine Chemicals, Piscataway, NJ), and the mixed leukocytes were separated through 43 and 53% discontinuous plasma-Percoll (Pharmacia) density gradients. The bottom interface yields a band of >95% pure PMNs, and the top interface yielded a band of mononuclear cells characteristically composed of 28% monocytes, 70% lymphocytes, and 2% PMNs. The mononuclear cells were pooled and washed twice with Krebs-Ringer phosphate buffer (pH 7.23) with 0.2% dextrose, minus calcium, plus 0.05% lipopolysaccharidefree human serum albumin (KRPD). The pellet was resuspended in KRPD buffer to a volume of 4-5 ml containing 2.5-3.5 X lo8 cells and further separated using a Beckman 52-21 centrifuge equipped with a strobe RPM JE-6B Elutriator Rotor (Beckman Instruments, Fullerton, CA) and the protocol outlined in Ref. 6. The cells were loaded at a flow rate of 10 ml/min onto the elutriator centrifuge head rotating at 2,500 t 10 rpm at 25OC. Fractions of 135 ml each were obtained at the following flow rates: 10 ml/min (loading fraction), 16 ml/min (fraction I), 21.5 ml/min (fraction 2), 28 ml/min (fraction 3), and a maximum purge rate (fraction 4). The cells from
1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 17, 2019.
LEUKOCYTE
AND PLATELET
each fraction were centrifuged at 275 g for 10 min and resuspended in KRPD buffer. A total cell count and nonspecific esterase stain were carried out on each fraction. Viability as determined by trypan blue dye exclusion was routinely >95%. Fraction 1 contained 97-100% pure lymphocytes with O-3% monocytes and represented -50% of the lymphocytes loaded onto the elutriator. Fraction 3 yielded 8896% pure monocytes with 58% lymphocytes and l-4% PMNs with no detectable platelet or erythrocyte (RBC) contamination and contained >75% of the monocytes initially loaded onto the elutriation system. The purified PMNs, monocytes, or lymphocytes were centrifuged at 275 g for 5 min and resuspended in 0.5 ml PPP. Cells were labeled either with 50-75 &i of “‘In in the presence of lo-* M tropolone at room temperature for 5 min (2-hydroxy-2,4,6-cycloheptatrienon, Fluka, Buch, FRG) or with 600 &i of 51Cr at 37°C for 30 min. The cells were then washed twice with PPP for infusion into the experimental animals. PMNs and monocytes prepared by these methods have been shown to behave in the circulation in a fashion similar to the native unisolated cells. The cells circulate with a half-life of 4 h for PMNs and 38 h for monocytes, the PMNs can be marginated and demarginated with the same specific activity, and both cells can localize to sites of inflammation (6, 9, 30). Platelets were isolated from 60 ml of rabbit blood anticoagulated with acid citrate dextrose by centrifuging the blood at 400 g for 10 min, decanting the plasma, and spinning the platelet-rich plasma at 800 g for 10 min (28). The platelets were then resuspended in buffer with 10% autologous rabbit plasma and labeled with 600 &i 51Cr for 60 min at 37°C or resuspended in buffer without plasma and labeled with 150 PC1 Yn-oxine for 15 min at room temperature. The platelets were centrifuged at 800 g for 10 min. The 51Cr-labeled platelets (‘Cr-platelets) were resuspended in 2 ml of buffer-lo% plasma for studies of first-pass extraction in the lungs while the ‘Yn-labeled platelets ( lllIn-platelets) were resuspended in 15 ml of buffer-lo% plasma for the lo-min retention studies (see below). Purity was assessed by hemocytometer counts and by histological examination of glutaraldehyde-fixed glycol methacrylate-embedded preparation of the platelet isolates. RBCs were radiolabeled with ggmTc (ggmTc-RBCs) using a Glucoscan kit (New England Nuclear Products, North Billeria, MA, model NRP-180) following the method of Gutkowski and Dworkin (8). Relative volumes of leukocytes. The volume of each leukocyte type was assessed using a cell counter (Sysmex E4000, Western Scientific), flow cytometry by forward scatter (Epics Profile, Coulter), and countercurrent centrifugation (cell elutriation), which separates cells on the basis of their size and density. Animal preparation. Twenty New Zealand White rabbits (3.3 t 0.08 kg) were anesthetized with intramuscular ketamine hydrochloride (25 mg/kg) and acepromazine maleate (2-3 mg/kg). The skin over the ventral neck was shaved and 1 ml xylocaine was injected subcutaneously. An endotracheal tube was inserted through a tracheos-
MARGINATION
1957
IN THE LUNG
tomy, and the animal breathed room air spontaneously throughout the experiment. A double-lumen catheter with separate injection ports was placed in the vena cava through the external jugular vein for the PMN, monocyte, and lymphocyte studies. When platelets were examined, a single-lumen catheter with one injection port was used. A single-lumen catheter was inserted into the aorta through the carotid artery for all four groups. Heparin (100 U/kg) was given after surgery, and blood samples were taken to determine circulating cell counts and arterial blood gases. Indicator-dilution studies. Indicator-dilution studies were performed as described elsewhere (5, 17, 18). Briefly, the ““Tc-RBCs were placed in one lumen of the double-lumen catheter with either Yn-PMNs, monocytes, or lymphocytes in the other. Homologous cells were used in the monocyte studies while both autologous and homologous cells were studied in the lymphocyte and PMN experiments as indicated in Table 1. These were flushed simultaneously into the central venous circulation with a bolus of 1.3 ml normal saline. Sequential blood samples (0.1-0.2 ml) were collected from the aortic catheter into preweighed tubes at 0.5-s intervals using a fraction collector. The blood samples were counted, and cell concentration vs. time curves were constructed using ggmTc and “‘In counts normalized to input counts per gram of blood. The cardiac output was determined by the indicator-dilution procedure (12, 31) using ggmTcRBCs as the indicator. The extraction of each blood cell type on the first passage through the pulmonary microvasculature was determined by comparing the areas under the leukocyte and RBC curves to the RBC peak (%extraction). Organ distribution of leukocytes. Within 15 s of the initial injection of ‘Yn-leukocytes, a second leukocyte type (either PMNs or lymphocytes) radiolabeled with 51Cr was injected into the central venous catheter in 13 of the 20 animals (Table 1). Additional ““Tc-RBCs were infused intra-arterially to measure blood volume. After 5 min, a peripheral blood sample was taken for cell counts. At 8-9 min, 1251-labeled macroaggregated albumin (5-50 pm, Charles E. Frosst, Kirkland, Quebec) was injected into the central venous catheter to mark pulmonary blood flow. At 10 min, a blood sample was taken for determination of cell counts, arterial blood gases, and radionuclide counts in the blood, and the animal’s heart TABLE
1. Type of blood cell infused in each experiment Cell
Types
Infused
PMNs PMNs + lymphocytes PMNs + monocytes Monocytes Lymphocytes Lymphocytes + monocytes Platelets (first-pass studies) Platelets (IO-min studies) Total number
Total
Autologous
Homologous
4 6 6 2 1
6 0 0 1
3 0 6 2 0
1
0
1
8 8 36
0 0 8
8 8 28
1
Values are number of experiments done with each cell type combinations of cell types (total) and are divided into homologous autologous cell infusions.
or or
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 17, 2019.
1958
LEUKOCYTE
AND
PLATELET
was stopped by intra-arterial injection of saturated potassium chloride. The chest and pericardial sac were rapidly opened, and a tie was placed around the base of the heart to maintain the pulmonary blood volume. The lungs were fixed in situ by intratracheal instillation of 7.5% glutaraldehyde in sodium phosphate buffer at 20 cmHaO pressure. The heart and lungs were removed en bloc and the lungs sectioned coronally into five slices of equal height where slice 1 was the lowest and slice 5 the highest in the gravitational field. The slices were cut into small pieces (total of 25 pieces for each animal) and placed in preweighed scintillation vials for gamma counting. The blood vessels supplying the liver were clamped and the liver removed without loss of blood. After sectioning, all liver parenchyma and blood were placed in scintillation vials. The spleen and one kidney were also removed and counted. Tissue and blood samples were counted in a Beckman 7000 gamma counter coupled to an Apple IIe computer with windows selected to maximize counts for each radioisotope while minimizing spillover into other channels. Corrections were made for overlap of radioisotope peaks by using pure reference samples of each isotope. Samples containing four isotopes were recounted after 2 wk to ensure accurate counting of Vr. The blood fractions from the indicator-dilution run were corrected for radiodecay to the time that the input was counted. The organ samples were corrected to the time that the first sample was placed in the gamma counter. Platelet studies. Separate experiments were necessary to determine platelet extraction on the first passage through the lungs and their distribution within organs 10 min after injection. 51Cr-platelets were used for firstpass studies (8 indicator-dilution studies in 3 rabbits). The organ distribution studies 10 min after platelet injection were performed in eight rabbits using platelets labeled with sufficient “‘In to allow the small marginated pool of platelets to be measured. The experimental protocol 1 ‘I* and the calculations were identical to the leukocyte stuales. Calculations. The regional blood volumes, regional blood flow, RBC transit time (RBC volume/blood flow), and the delivery, retention, and exch .ange rates of radiolabeled leukocytes were calcul ated as previously described (5, 12, 18). Statistical analysis. Calculations are expressed as means t SE. All data were analyzed using the nonparametric Kruskal-Wallis analysis of variance with multiple comparisons based on the ranks (4). The relationship of both percent retention and exchange rate with RBC transit time was examined for each cell type, and the different cell types were compared. After logarithmic transformations of the retentions, exchange rates, and RBC transit times, the retentions and exchange rates were grouped by RBC transit time intervals. The relationship between retention or exchange rate with transit time was evaluated using a nested analysis of variance. The change in retention or exchange rate with increasing RBC transit time was examined within each cell type, and the cell types were compared.
MARGINATION
IN
THE
LUNG
RESULTS Isolated blood cell preparations. The cell purity was 94 + 0.6% for PMNs, 98 t 0.9% for lymphocytes, and 89 t 0.7% for monocytes. No shape changes suggestive of activation were observed in either PMNs or monocytes. The specific activity was 0.071 t 0.020 counts/min (cpm)/PMN, 0.21 & 0.07 cpm/monocyte, and 0.081 t 0.002 cpm/lymphocyte. The cell concentration of the injected sample was 33.5 t 4.7 X lo6 PMNs/ml, 12.5 t 1.8 x lo6 monocytes/ml, and 17.5 t 4.0 X lo6 lymphocytes/ml. Previous work from our laboratories has shown that PMNs and monocytes prepared using these methods retain their functional abilities when tested in vitro and in vivo (6, 9, 30). The isolated platelets contained no white cells and
D. ENGLISH, J. C. HOGG
R. P. GIE,
The University of British Columbia Pulmonary Research Laboratory, Vancouver, British Columbia V6Z 1 Y6, Canada; and Departments of Medicine and Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Service of Medicine, Denver Veterans Administration Hospital, and Departments of Pathology and Medicine, Section of Pulmonary Diseases, University of Colorado School of Medicine, Denver, Colorado 80206
DOERSCHUK, C. M., G. P. DOWNEY, D. E. DOHERTY,D. ENGLISH, R. P. GIE, M. OHGAMI, G. S. WORTHEN,P. M. HENSON, AND J. C. HOGG. Leukocyte and platelet margination within microvasculuture of rabbit lungs. J. Appl. Physiol. 68(5): 1956-1961, 1990.-These studiescompare the behavior of radiolabeled neutrophils, monocytes, lymphocytes, and platelets during their first passthrough the pulmonary circulation after a central venous injection and their distribution within the circulation 10min later. Their first passthrough the pulmonary circulation was comparedwith erythrocytes (RBCs) using the indicator-dilution technique, and their recovery within the circulation of the lung and other organswas determined at 10 min by counting the radioisotopesin eachorgan. The extraction of each cell relative to RBCs during the first passthrough the lung correlated with cell size in that the neutrophils (volume 107-140fl) showed97.6 t 0.6% extraction, monocytes(volume 80-105 fl) showed91.4t 1.7%extraction, lymphocytes (volume 36-75 fl) showed80.1 2 4.4% extraction, and platelets (volume 4-7 fl) showed33.1 t 3.9% extraction. After 10 min of circulation, the proportion of injected cells remaining in the lung was similar for neutrophils and monocytes (27.4 k 1.8 vs. 31.4 t 1.6%) but lower for lymphocytes (18.6 k 2.9%) and platelets (3.1 t 0.5%). All of the leukocytes were found to have a substantial marginatedpool within the lung, whereasthe platelets did not. The exchangebetweenthe circulating and marginated pools of leukocytes in the lung was related to blood velocity, with the least retention occurring in lung regionswith shortest RBC transit times. We conclude that cell size is a major factor determining the time that cells will be delayed by the pulmonary microvasculature. However, other factors such as the cells’ deformability and their interaction with the endothelium may alsobe important.
neutrophils; monocytes; lymphocytes; platelets; erythrocyte transit time; pulmonary marginated leukocyte pool
THE PULMONARY capillary
bed is made up of a large number of short interconnected segments that range in size from 2 to 12 m in diameter so that some are narrow enough to restrict the passage of the circulating cells (1, ll,l3). Although erythrocytes and leukocytes have similar diameters, the erythrocytes can move through these restrictions much more easily because they are more deformable (3,11,13,16,22,23,25). The multisegmented nature of the pulmonary capillary bed allows the eryth1956
0161-7567/90 $1.50 Copyright
0
rocytes to stream around the neutrophils, which are delayed in these restrictive segments. This concentrates the neutrophils with respect to erythrocytes, which accounts for the large marginated pool of neutrophils in the pulmonary capillary bed (5, 11, 13). The purpose of this study was to extend our previous observations on neutrophils to lymphocytes, monocytes, and platelets to determine if these cells also marginate within the lung microvessels. METHODS Isolation and labeling of leukocytes. The methods for isolating neutrophils (PMNs), monocytes, lymphocytes, and platelets used in this study have been fully described elsewhere (6, 9, 28, 30). Briefly, titrated whole blood, collected by sterile puncture of the central ear artery of unanesthetized New Zealand White rabbits, was centrifuged at 300 g for 20 min. The platelet-rich plasma was aspirated and centrifuged at 2,500 g for 20 min to yield platelet-poor plasma (PPP). The remaining cells were sedimented with dextran (molecular wt 500,000, Pharmacia Fine Chemicals, Piscataway, NJ), and the mixed leukocytes were separated through 43 and 53% discontinuous plasma-Percoll (Pharmacia) density gradients. The bottom interface yields a band of >95% pure PMNs, and the top interface yielded a band of mononuclear cells characteristically composed of 28% monocytes, 70% lymphocytes, and 2% PMNs. The mononuclear cells were pooled and washed twice with Krebs-Ringer phosphate buffer (pH 7.23) with 0.2% dextrose, minus calcium, plus 0.05% lipopolysaccharidefree human serum albumin (KRPD). The pellet was resuspended in KRPD buffer to a volume of 4-5 ml containing 2.5-3.5 X lo8 cells and further separated using a Beckman 52-21 centrifuge equipped with a strobe RPM JE-6B Elutriator Rotor (Beckman Instruments, Fullerton, CA) and the protocol outlined in Ref. 6. The cells were loaded at a flow rate of 10 ml/min onto the elutriator centrifuge head rotating at 2,500 t 10 rpm at 25OC. Fractions of 135 ml each were obtained at the following flow rates: 10 ml/min (loading fraction), 16 ml/min (fraction I), 21.5 ml/min (fraction 2), 28 ml/min (fraction 3), and a maximum purge rate (fraction 4). The cells from
1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 17, 2019.
LEUKOCYTE
AND PLATELET
each fraction were centrifuged at 275 g for 10 min and resuspended in KRPD buffer. A total cell count and nonspecific esterase stain were carried out on each fraction. Viability as determined by trypan blue dye exclusion was routinely >95%. Fraction 1 contained 97-100% pure lymphocytes with O-3% monocytes and represented -50% of the lymphocytes loaded onto the elutriator. Fraction 3 yielded 8896% pure monocytes with 58% lymphocytes and l-4% PMNs with no detectable platelet or erythrocyte (RBC) contamination and contained >75% of the monocytes initially loaded onto the elutriation system. The purified PMNs, monocytes, or lymphocytes were centrifuged at 275 g for 5 min and resuspended in 0.5 ml PPP. Cells were labeled either with 50-75 &i of “‘In in the presence of lo-* M tropolone at room temperature for 5 min (2-hydroxy-2,4,6-cycloheptatrienon, Fluka, Buch, FRG) or with 600 &i of 51Cr at 37°C for 30 min. The cells were then washed twice with PPP for infusion into the experimental animals. PMNs and monocytes prepared by these methods have been shown to behave in the circulation in a fashion similar to the native unisolated cells. The cells circulate with a half-life of 4 h for PMNs and 38 h for monocytes, the PMNs can be marginated and demarginated with the same specific activity, and both cells can localize to sites of inflammation (6, 9, 30). Platelets were isolated from 60 ml of rabbit blood anticoagulated with acid citrate dextrose by centrifuging the blood at 400 g for 10 min, decanting the plasma, and spinning the platelet-rich plasma at 800 g for 10 min (28). The platelets were then resuspended in buffer with 10% autologous rabbit plasma and labeled with 600 &i 51Cr for 60 min at 37°C or resuspended in buffer without plasma and labeled with 150 PC1 Yn-oxine for 15 min at room temperature. The platelets were centrifuged at 800 g for 10 min. The 51Cr-labeled platelets (‘Cr-platelets) were resuspended in 2 ml of buffer-lo% plasma for studies of first-pass extraction in the lungs while the ‘Yn-labeled platelets ( lllIn-platelets) were resuspended in 15 ml of buffer-lo% plasma for the lo-min retention studies (see below). Purity was assessed by hemocytometer counts and by histological examination of glutaraldehyde-fixed glycol methacrylate-embedded preparation of the platelet isolates. RBCs were radiolabeled with ggmTc (ggmTc-RBCs) using a Glucoscan kit (New England Nuclear Products, North Billeria, MA, model NRP-180) following the method of Gutkowski and Dworkin (8). Relative volumes of leukocytes. The volume of each leukocyte type was assessed using a cell counter (Sysmex E4000, Western Scientific), flow cytometry by forward scatter (Epics Profile, Coulter), and countercurrent centrifugation (cell elutriation), which separates cells on the basis of their size and density. Animal preparation. Twenty New Zealand White rabbits (3.3 t 0.08 kg) were anesthetized with intramuscular ketamine hydrochloride (25 mg/kg) and acepromazine maleate (2-3 mg/kg). The skin over the ventral neck was shaved and 1 ml xylocaine was injected subcutaneously. An endotracheal tube was inserted through a tracheos-
MARGINATION
1957
IN THE LUNG
tomy, and the animal breathed room air spontaneously throughout the experiment. A double-lumen catheter with separate injection ports was placed in the vena cava through the external jugular vein for the PMN, monocyte, and lymphocyte studies. When platelets were examined, a single-lumen catheter with one injection port was used. A single-lumen catheter was inserted into the aorta through the carotid artery for all four groups. Heparin (100 U/kg) was given after surgery, and blood samples were taken to determine circulating cell counts and arterial blood gases. Indicator-dilution studies. Indicator-dilution studies were performed as described elsewhere (5, 17, 18). Briefly, the ““Tc-RBCs were placed in one lumen of the double-lumen catheter with either Yn-PMNs, monocytes, or lymphocytes in the other. Homologous cells were used in the monocyte studies while both autologous and homologous cells were studied in the lymphocyte and PMN experiments as indicated in Table 1. These were flushed simultaneously into the central venous circulation with a bolus of 1.3 ml normal saline. Sequential blood samples (0.1-0.2 ml) were collected from the aortic catheter into preweighed tubes at 0.5-s intervals using a fraction collector. The blood samples were counted, and cell concentration vs. time curves were constructed using ggmTc and “‘In counts normalized to input counts per gram of blood. The cardiac output was determined by the indicator-dilution procedure (12, 31) using ggmTcRBCs as the indicator. The extraction of each blood cell type on the first passage through the pulmonary microvasculature was determined by comparing the areas under the leukocyte and RBC curves to the RBC peak (%extraction). Organ distribution of leukocytes. Within 15 s of the initial injection of ‘Yn-leukocytes, a second leukocyte type (either PMNs or lymphocytes) radiolabeled with 51Cr was injected into the central venous catheter in 13 of the 20 animals (Table 1). Additional ““Tc-RBCs were infused intra-arterially to measure blood volume. After 5 min, a peripheral blood sample was taken for cell counts. At 8-9 min, 1251-labeled macroaggregated albumin (5-50 pm, Charles E. Frosst, Kirkland, Quebec) was injected into the central venous catheter to mark pulmonary blood flow. At 10 min, a blood sample was taken for determination of cell counts, arterial blood gases, and radionuclide counts in the blood, and the animal’s heart TABLE
1. Type of blood cell infused in each experiment Cell
Types
Infused
PMNs PMNs + lymphocytes PMNs + monocytes Monocytes Lymphocytes Lymphocytes + monocytes Platelets (first-pass studies) Platelets (IO-min studies) Total number
Total
Autologous
Homologous
4 6 6 2 1
6 0 0 1
3 0 6 2 0
1
0
1
8 8 36
0 0 8
8 8 28
1
Values are number of experiments done with each cell type combinations of cell types (total) and are divided into homologous autologous cell infusions.
or or
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 17, 2019.
1958
LEUKOCYTE
AND
PLATELET
was stopped by intra-arterial injection of saturated potassium chloride. The chest and pericardial sac were rapidly opened, and a tie was placed around the base of the heart to maintain the pulmonary blood volume. The lungs were fixed in situ by intratracheal instillation of 7.5% glutaraldehyde in sodium phosphate buffer at 20 cmHaO pressure. The heart and lungs were removed en bloc and the lungs sectioned coronally into five slices of equal height where slice 1 was the lowest and slice 5 the highest in the gravitational field. The slices were cut into small pieces (total of 25 pieces for each animal) and placed in preweighed scintillation vials for gamma counting. The blood vessels supplying the liver were clamped and the liver removed without loss of blood. After sectioning, all liver parenchyma and blood were placed in scintillation vials. The spleen and one kidney were also removed and counted. Tissue and blood samples were counted in a Beckman 7000 gamma counter coupled to an Apple IIe computer with windows selected to maximize counts for each radioisotope while minimizing spillover into other channels. Corrections were made for overlap of radioisotope peaks by using pure reference samples of each isotope. Samples containing four isotopes were recounted after 2 wk to ensure accurate counting of Vr. The blood fractions from the indicator-dilution run were corrected for radiodecay to the time that the input was counted. The organ samples were corrected to the time that the first sample was placed in the gamma counter. Platelet studies. Separate experiments were necessary to determine platelet extraction on the first passage through the lungs and their distribution within organs 10 min after injection. 51Cr-platelets were used for firstpass studies (8 indicator-dilution studies in 3 rabbits). The organ distribution studies 10 min after platelet injection were performed in eight rabbits using platelets labeled with sufficient “‘In to allow the small marginated pool of platelets to be measured. The experimental protocol 1 ‘I* and the calculations were identical to the leukocyte stuales. Calculations. The regional blood volumes, regional blood flow, RBC transit time (RBC volume/blood flow), and the delivery, retention, and exch .ange rates of radiolabeled leukocytes were calcul ated as previously described (5, 12, 18). Statistical analysis. Calculations are expressed as means t SE. All data were analyzed using the nonparametric Kruskal-Wallis analysis of variance with multiple comparisons based on the ranks (4). The relationship of both percent retention and exchange rate with RBC transit time was examined for each cell type, and the different cell types were compared. After logarithmic transformations of the retentions, exchange rates, and RBC transit times, the retentions and exchange rates were grouped by RBC transit time intervals. The relationship between retention or exchange rate with transit time was evaluated using a nested analysis of variance. The change in retention or exchange rate with increasing RBC transit time was examined within each cell type, and the cell types were compared.
MARGINATION
IN
THE
LUNG
RESULTS Isolated blood cell preparations. The cell purity was 94 + 0.6% for PMNs, 98 t 0.9% for lymphocytes, and 89 t 0.7% for monocytes. No shape changes suggestive of activation were observed in either PMNs or monocytes. The specific activity was 0.071 t 0.020 counts/min (cpm)/PMN, 0.21 & 0.07 cpm/monocyte, and 0.081 t 0.002 cpm/lymphocyte. The cell concentration of the injected sample was 33.5 t 4.7 X lo6 PMNs/ml, 12.5 t 1.8 x lo6 monocytes/ml, and 17.5 t 4.0 X lo6 lymphocytes/ml. Previous work from our laboratories has shown that PMNs and monocytes prepared using these methods retain their functional abilities when tested in vitro and in vivo (6, 9, 30). The isolated platelets contained no white cells and
