Development of pulmonary intravascular function in newborn lambs KIM E. LONGWORTH, ANNE AND NORMAN C. STAUB

M. WESTGATE,

MICHAEL

macrophage

K. GRADY,

JAY Y. WESTCOTT,

Cardiovascular Research Institute and Department of Physiology, University of California, Sun Francisco, California 94143; and Pulmonary Department, University of Colorado Health Sciences Center, Denver, Colorado 80262

lung defense and, consequently, lung inflammation and injury. Although the presence of intravascular macrophages is nearly always associated with enhanced pulmonary vascular reactivity, it has not been definitively established that pulmonary intravascular macrophages are the cause of pulmonary hypertension or other components of the inflammatory response. To demonstrate cause and effect, Koch’s postulates must be confirmed for the intravascular macrophages (12). The first postulate, that the cells are present whenever the responses occur, has been demonstrated in sheep, goats, calves, and pigs (13). The second, that cells can be isolated and shown to respond to specific stimuli, has been explored in a preliminary way by Bertram et al. (3), Chitko-McKown et al. (6), and Fowler et al. (11). They have shown that cells with the characteristics of intravascular macrophages isolated from the lungs of pigs and studied in vitro phagocytize part.icles such as iron, asbestos, carbon, and bacteria and release products of arachidonic acid metabolism, cytokines, and other potential mediators of inflammat,ion. The third postulate, that absence or inhibition of the cells causes the responses to disappear and that restoration or appearance of the cells causes the responses to occur, is the subject of our study. We have circumvented the macrophages radioactive liposomes;Monastral blue; mononuclearphagocyte the difficulty of removing or inhibiting and then restoring them by studying newborn lambs system; pulmonary edema; pulmonary hypertension; thromthat, as our survey shows (see below), only develop intraboxane production; sheep vascular macrophages after birth. Lambs follow the same pattern as piglets, which are born with few pulmonary intravascular macrophages but develop a large populaPULMONARY INTRAVASCULAR macrophages (hereafter tion within several days (29, 30). Thus we tested Koch’s referred to as macrophages or intravascular macrophages) are a resident population of mononuclear cells in third postulate by measuring the pulmonary vascular recertain species of mammals, principally those in the activity of the lambs to foreign particles shortly after birth, when few macrophages are present, and after 2 wk, order Artiodactyla (5,19,22,23). In those mammals with a large population of intravascular macrophages, the when large numbers are resident in the pulmonary capillaries. lung is the principal site of clearance of particles injected We asked the following questions. In lambs, does pulintravenously (8, 13, 14, 17, 22, 25, 27). In addition, the interaction between these resident macrophages and for- monary vascular reactivity to foreign particles increase eign blood-borne particles is usually associated with in- from birth to 2 wk of age? Does a pulmonary vasoconcreased pulmonary vascular reactivity (2, 13, 14, 27). strictor that macrophages are capable of releasing cause Studies using isolated mononuclear phagocytes washed such an increase? Is the increase in reactivity associated with an increase in the uptake of particles by macroout of the pulmonary vessels elf pigs (putative macrophages) have shown that the cells are capable of releas- phages in the pulmonary capillaries? ing pulmonary vasoconstrictors (3, 4). Thus, in species To answer the first two questions, we tested the pulmothat have these cells, they may play an important role in nary vascular reactivity of newborn (l-3 days old) and LONGWORTH,KIM &ANNE M. WESTGATE, MICHAEL K. ANDNORMAN C. STAUB.Development of pulmonary intravascular macrophuge function in newborn lambs. J. Appl. Physiol. 7316): 2608-2615, 1992.-We sought to determine whether pulmonary intravascular macrophagesare involved in pulmonary vascular sensitivity to intravenously injected particles in sheep.We estimated that newborn lambshave few of these macrophagesat birth but develop a 10-fold greater density within 2 wk. Awake, chronically instrumented newborn lambs showed no change in pulmonary vascular driving pressure(pulmonary arterial minus left atria1 pressure) after injection of either liposomes [2 t 3 (SD) cmH,O; n = 51 or Monastral blue particles (3 t 2 cmH,O; n = 6) and showedno net pulmonary production of thromboxane B,, the stable metabolite of the vasoconstrictor thromboxane A,. In contrast, five of those lambs 2 wk later showedboth an increasein pulmonary vascular driving pressureafter injection of liposomesand Monastral blue (20 t 16 and 25 k 15 cmH,O, respectively; P < 0.05) and net pulmonary production of thromboxane B, (171 & 103 and 429 t 419 pg/ml plasma, respectively; P c 0.05). Older lambs (n = 5) had higher pulmonary uptakes than newborn lambs (n = 6) of radioactive liposomes(47 t 13 vs. 12 t 10%; P < 0.01) and Monastral blue (53 t 6 vs. 21 t 10%; P < 0.05). We conclude that pulmonary intravascular macrophagesare responsiblefor the sensitivity of sheepto intravenous foreign particles and are essentialfor a cascadeof processesleading to microvascular injury.

GRADY,JAYY.WESTCOTT,

2608

0161-75671'92

$2.00 Copyright

0

1992the

American

Physiological

Society

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2-wk-old lambs to two foreign particles, radioactively labeled liposomes and Monastral blue pigment. We measured hemodynamic variables and production of thromboxane B,, a stable metabolite of thromboxane A,; the latter is a potent vasoconstrictor produced by the cyclooxygenase pathway of arachidonic acid metabolism. To answer the third question, we measured uptake of particles in the lungs of lambs of both ages. Our preliminary quantitative histological data confirm that although newborn lambs have few intravascular macrophages, 2-wk-old lambs have a substantial population. Our physiological data show that pulmonary vascular reactivity to particles increases with age, concomitant with an increase in uptake of particles by the lungs.

tom borders of the lattice. We analyzed three sections (each from a different block) per lamb until 400 septal tissue points had been counted. We used the ratio of points on tissue to total area of the field to estimate alveolar septal tissue surface area, and then we calculated the number of macrophages per square millimeter of alveolar septal tissue. Materials. To test for the presence and activity of pulmonary intravascular macrophages in lambs l-3 and l316 days after birth, we prepared radioactive liposomes and Monastral blue pigment as intravenous injectates. Bilayer liposomes composed of phosphatidylcholine (Avanti Polar Lipids), cholesterol (Sigma Chemical), and phosphatidylserine (Avanti Polar Lipids), combined in the molar ratio 6:4:1, were prepared by reverse-phase evaporation (14). After manufacture they were filtered METHODS through a l-pm polycarbonate filter. The liposomes were Histological analysis. To confirm that the observations labeled by incubation with a Yndium chloride-tropoof Winkler and Cheville (29, 30) on the development of lone complex (lllIn from Amersham). After incubation, pulmonary intravascular macrophages in piglets were free “‘In was removed by repeated centrifugation and also true for lambs, we estimated the macrophage popularinsing. The final concentration of liposomes was 10 tions in two newborn and two 2-wk-old lambs that had pmol total lipids/ml in phosphate-buffered saline. A been intravenously injected with 1 ml/kg of a 1% suspenfixed dose of 10 pmol in 1 ml saline was used to test the sion of Monastral blue. We fixed the lungs by vascular hemodynamic response of each lamb. perfusion to best visualize the intravascular macroMonastral blue B (Sigma Chemical) is a copper phthaphages; this method distends the capillaries and removes locyanine pigment. It is supplied as *a 3% aqueous suspenmost of the blood leukocytes but not the intravascular sion of particles of w-45-60 nm diameter. We prepared a macrophages, which remain attached to the endothelial 1% suspension of Monastral blue by diluting this stock surface. After fixation, we randomly sampled the lower suspension in normal saline. A dose of 0.5 ml/kg was lobe of the right lung for light microscopy. We cut the used to test the hemodynamic response of each lamb. lung into six to eight l-cm-thick slices, placed a grid with To determine whether the pulmonary vasculature in l-cm2 squares over the slices, and, using randomly cho- newborn lambs was capable of responding to thromboxsen pairs of numbers to identify coordinates, cut one samane in the blood, a thromboxane mimetic was prepared ple from each of four randomly chosen slices. These four for intravenous injection. The mimetic, U-46619 (Upsample blocks (10 X 5 X 2 mm) were dehydrated and john, Kalamazoo, MI), was diluted in ethanol to a conembedded in plastic. We cut 2-pm-thick sections from centration of 2 pglpl. We gave 2 pg U-46619 in 1 ml saline each, mounted the sections on glass slides, and stained as the test dose. them with toluidine blue. The color of this stain is blueNone of the carriers for the injectates produced meapurple and is easily distinguished from the bright sky surable hemodynamic or other responses in any lambs blue of Monastral blue. when intravenously injected. We estimated the number of macrophages per crossAnimals. We used 11 newborn lambs; 10 were obtained sectional area of alveolar septal tissue, excluding air from ewes that underwent normal parturition after norspace, by modifying standard point-counting methods mal gestation (-145 days), and 1 was obtained by cae(28). Expressing the number of macrophages per unit sarean section of the ewe at 143 days. The lambs were area septal tissue avoids the need to account for possible allowed to stay with their mothers for 6-12 h before prepdifferences in inflation or alveolar size among the lungs. aration. We viewed the lung tissue sections with a light microSurgical preparation. We prepared the lambs for the scope (Olympus BH-2) at ~750 and superimposed a co- experiments by surgical implantation of catheters in 10 herent square lattice (100 points; 17,956 pm2 area) by lambs after birth and in the 1 lamb delivered by caesarinserting a graticule into the eyepiece. It was usually, but ean section at delivery. After anesthetizing each lamb not always, easy to distinguish intravascular macrowith an intramuscular injection of ketamine hydrochlophages in these sections, so we used the presence or ab- ride (10 mg/kg), we inserted heparin-coated (TDMAC, sence of Monastral blue to definitively identify a cell as a Polysciences, Warrington, PA) Tygon (polyvinylidine macrophage; thus, only cells containing Monastral blue chloride) catheters into a vein and artery of the distal were counted as macrophages. We analyzed nonoverlaphindlimb using a local anesthetic (lidocaine) and adping fields; in each field we recorded the number of lat- vanced them until the lengths inserted indicated that the tice points falling on septal tissue (structures 10.1 mm) tips were situated in the inferior vena cava and the deand excluded points falling on air space. We then SUF- scending aorta, respectively. Then we fully anesthetized veyed the field and counted the total number of intravasthe lambs using halothane (4-5%) administered briefly cular macrophages containing Monastral blue visible through a face mask and after endotracheal intubation within it. Excluded from the counting were macrophages connected them to a piston pump ventilator (Harvard or points on septal tissue that touched the right or bot- Apparatus). Anesthesia and ventilation were maintained Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 10, 2019.

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with 0251.25% halothane at 15 ml/kg tidal volume in a gas mixture of 60% air-40% 0,. Arterial blood gases, pH, and heart rate were monitored throughout the surgery. Through a left thoracotomy via the third or fourth intercostal space we ligated the ductus arteriosus and then placed Tygon catheters in the pulmonary artery and left atrium for pressure measurements and a 3.5-Fr thermistor (Edwards Laboratories) in the pulmonary artery for cardiac output measurements. All three catheters and the thermistor exited the thorax through the sixth intercostal space and tunneled under the skin to approximately the eleventh rib. The chest was closed, and the lamb was allowed to awaken before returning to its mother. For up to 2 wk we daily gave the lambs intravenous injections of antibiotics (100,000 U/kg penicillin and 2.3 mg/kg gentamicin) and maintained the catheters by withdrawing dead space contents and refilling with heparin. ProtocoZ. The 11 lambs were placed in two groups. We studied one group of six lambs l-3 days after birth for their hemodynamic responses to intravenous injections of radioactive liposomes, Monastral blue pigment, and the thromboxane mimetic. We then anesthetized each lamb, opened the thorax, fixed the right lung for histology, and removed the left lung together with the liver, spleen, and 10 ml of blood for analysis of radioactivity and copper. We studied the other group of five lambs l-3 days after birth and again after 2 wk (13-16 days). On days 1-3, we tested their hemodynamic responses to injections of radioactive liposomes and the thromboxane mimetic. On days 13-16, we tested them again using the same injectates, as well as Monastral blue. At the end of the experiments on each lamb one lung was fixed and other tissues were removed for analysis as described above. Thus there are both paired and unpaired data on 11 lambs l-3 days old and on 5 lambs 2 wk old; the latter are the “paired subset.” For each experiment, we placed the awake lamb in a sling and prepared the catheters for pressure measurements, blood samples, and injection of test substances. After a 15-min baseline recording, we injected test substances as a bolus via the hindlimb venous catheter and continuously recorded pressure changes. Thirty to 60 min after the lamb’s pulmonary arterial pressure returned to baseline, or after 15 min if no response occurred, we injected the next substance. We gave two doses each of liposomes or Monastral blue; during the first we recorded the complete time course of all pressure changes, and during the second we drew pulmonary arterial and systemic arterial blood samples at the time when the peak in pulmonary arterial pressure had occurred during the first dose. On the final day of experiments on a lamb, we deeply anesthetized the lamb with halothane, gave it heparin (1,000 U/kg), placed it supine, and opened the chest via a sternotomy. After clamping the vessels and airways to the left lung, we removed the lung from the chest and then fixed the right lung for histology (to be used in a later study). Then we inserted a large bore catheter in the right pulmonary artery and opened the left atrium. At a pressure of 20 cmH,O, we perfused the vascular bed with

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saline until the left atria1 effluent was clear and continued perfusion with 2-3 liters of fixative (2.4% glutaraldehyde and 1% paraformaldehyde in Millonig’s phosphate buffer; pH 7.45, 320 mosmol/kgH,O). Airway pressure was held constant at 10 cmH,O. After the right lung was fixed and removed from the chest, we removed the liver, spleen, and some rib bone marrow for analysis and weighed all the organs. Recordings. We measured pulmonary arterial, left atrial, and systemic arterial pressures by connecting the respective catheters to lightweight pressure transducers (Medex) and a direct writing polygraph (Grass model 7), with reference at midchest level. We measured cardiac output by thermal dilution (5 ml iced saline) using a cardiac output computer (KMA model 3500). To avoid interference with pulmonary arterial pressure recording after injection, we measured cardiac output before each injection and after pulmonary arterial pressure had returned to baseline values. However, to confirm that the increase in pulmonary arterial pressure was due to an increase in pulmonary vascular resistance, in a few experiments we measured cardiac output after sampling for thromboxane but while pulmonary arterial pressure was still elevated or gave an additional injection of test substance and measured cardiac output at the peak of the pressure response. Blood and organ analysis. At the beginning and end of each experiment, we measured blood gases and pH using a blood gas analyzer (model 158, Corning). The thermistor in the pulmonary artery provided the core temperature of the lamb for correction of the blood gases. After withdrawal of catheter dead space, we drew simult!aneous mixed venous and systemic arterial blood samples (2 ml) into plastic unheparinized syringes for thromboxane B, analysis. The blood was immediately placed into iced test tubes containing 2 mg EDTA and 50 pg indomethacin and was centrifuged at 10°C for 20 min. The supernatant was removed, placed in a polypropylene test tube, and stored at -7OOC. The samples were analyzed for thromboxane B,, a stable metabolite of thromboxane A,, by sensitive enzyme immunoassay (15), using an antibody provided by Dr. Jacques Maclouf (INSERM, Paris, France). The test can detect a concentration as low as 15 pg/ml plasma. We homogenized the various tissues for analysis. To determine relative amounts of lllIn-labeled liposomes in lung, liver, spleen, blood, and bone marrow, we measured the radioactivity of duplicate 0.5-g samples of each in a well-type counter (Packard MINAXI Gamma 500 series). The mean radioactivity recovered from the tissues sampled was 89 t 10 (SD)% of the injected dose. We assumed blood volume was 7% and bone marrow was 2% of body mass to calculate the total radioactivity of each. To determine relative amounts of Monastral blue in the lungs, we measured copper concentration in the homogenized tissue (10). Weighed tissue samples of - 1 g were dissolved in 5 ml concentrated (15.7 N) nitric acid and slowly boiled until the tissue was dissolved, and the resulting clear liquid was evaporated (-24 h). Then the residue was mixed with exactly 5 ml nitric acid, and the solution was measured in an atomic absorption spectrophotometer (model 151, Instrumentation Laboratories,

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TABLE 1. Histological data showing increase with age in population of pulmonary intravascular macrophages containing Monastral blue Lamb 1

2

Days

l-3

232270 84f24

Lamb 3 4

Values are means +- SD. No. of macrophages alveolar septal tissue (excluding air space), each age on 3 lung sections each.

Days

12-16

1,566?263 1,459&485 is expressed per mm2 of estimated on 2 lambs at

Lexington, MA) for absorbance at 324.7 nm. We zeroed the instrument with deionized water or the 1 N nitric acid in deionized water that was used to prepare the samples and standard solutions. Standard solutions were prepared by volumetric dilution of a stock standard of 1.0000 g copper metal dissolved in 65 ml concentrated nitric acid and diluted to 1.000 liter with distilled, deionized water. We obtained control samples from four lambs not injected with Monastral blue to estimate natural copper concentrations in lung tissues. We subtracted the mean concentration 11.03 k 0.44 (SD) pg Cu/g wet lung] from the concentration in each experimental lamb to calculate net copper concentrations. Statistics. We used a paired t test or one-way analysis of variance to make comparisons between ages when variances were equal. Where variances were unequal between ages, we used a Wilcoxon signed-ranks test for paired observations and a Wilcoxon two-sample test for unpaired observations (18). Analysis of variance was done using the Statpro statistics program (Penton Software). RESULTS

Preliminary histological data. The number of macrophages containing Monastral blue per square millimeter alveolar septal tissue was w lo-fold higher (158 vs. 1,513/ mm2) in older than in younger lambs (Table 1, Fig. 1). The minimum increase measured was sixfold, from 232 + 70 to 1,459 + 485 (SD) macrophages/mm2. Physical condition. All the lambs were in good physical

FIG. 1. Monastral old lambs (B). Higher

blue particles (arrowheads) density of macrophages

in pulmonary in older lambs

intravascular is obvious.

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condition on the day of the study. The five lambs in the paired subset remained healthy during the 2 wk before the final experiment; they all gained weight and maintained a normal body temperature (395°C) (Table 2). Although we took the smallest possible blood sample volumes for analysis, hematocrit decreased during the 2 wk [32 f 5 (SD) vs. 23 + 6%]; however, the blood gases and pH remained within normal limits for newborn lambs. We observed the lambs’ behavior each day; they were active and feeding well. The initial data on the other six lambs were similar to those in the paired subset (body weight, 5.0 + 0.8 kg; hematocrit, 30 f 4%; body temperature, 39.5 f 0.4”C; arterial PO,, 82 f 11 Torr; arterial Pco,, 43 + 4 Torr; arterial pH, 7.4 * 0.1). Response to liposomes. Pulmonary vascular reactivity to liposomes increased with age (Table 3). After injection of 10 pmol liposomes on days l-3, the five lambs in the paired subset showed a slight increase in pulmonary arterial pressure and no change in left atria1 pressure. Consequently, pulmonary vascular driving pressure (pulmonary arterial minus left atria1 pressure) only increased by 2 -t 2 cmH,O (3 + 4 cmH,O for all 11 lambs). Although both pulmonary and systemic arterial concentrations of thromboxane increased after injection, pulmonary arterial thromboxane concentrations increased more; thus net pulmonary production of thromboxane was -50 -t 141 pg/ml plasma. This negative value could indicate that more thromboxane was metabolized than produced in the lung. In contrast, on days 13-16 the five lambs in the paired subset showed a substantial increase in pulmonary arterial pressure and no change in left atria1 pressure after injection. Consequently, pulmonary vascular driving pressure increased by 20 f 16 cmH,O, a sevenfold greater increase than at the younger age. In addition, net pulmonary thromboxane production was always positive (171 + 103 pg/ml plasma). Uptake of liposomes by the lung also increased with age, from 12 + 10% at l-3 days to 47 * 13% at 13-16 days (Fig. 2). In contrast, uptake by the liver decreased from 60 f 13% at l-3 days to 32 * 9% at 13-16 days. Uptake of liposomes by the spleen and marrow did not change with age; the blood concentration was 2 and 4%, respectively (P < 0.05).

macrophages

of newborn

(A) and 2-wk-

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TABLE 2. Baseline physiulugical variables for newborn lambs in the paired study Days 1-3

Body weight, kg Hematocrit, % Body temperature, “C Arterial PO,, Ton Arterial Pco~, Torr Arterial pH

4.9+0.7 32t5

39.5t0.5 77tl2 44t5 7.4t0.0

Days 12-16

8.4*1*1* 22.8+6*

39.7d.6 SW11 45*3 7.4IkO.Q

Values are group means + SD; IZ= 5 lambs. Blood gases and pH are corrected to pulmonary arterial temperature. * Significantly different from days I-3 (P < 0.05).

Response to Munastrai! blue. The lambs’ pulmonary hemodynamic responses to Monastral blue at both ages (unpaired data) were similar to their responses to liposomes (Table 4). After injection of 0.5 ml/kg Monastral blue on days l-3, six lambs showed a slight increase in pulmonary arterial pressure, no change in left atria1 pressure, and a slight increase in pulmonary vascular driving pressure (3 t 2 cmH,O). Both pulmonary and arterial thromboxane concentrations increased slightly and by about the same amount; net pulmonary production of thromboxane was essentially zero. In contrast, on days 13-16 the lambs showed a substantial increase in pulmonary arterial pressure, no change in left atria1 pressure, and, therefore, a large increase in pulmonary vascular driving pressure (25 -t 15 cmH,O), an eightfold greater change than in the younger lambs. In addition, net pulmonary thromboxane production was positive (429 t 419 pglml plasma). Monastral blue concentrations in lung tissues increased almost three times with age. Uptake of Monastral blue in the lungs, calculated as the percent of injected dose, increased 2.5 times with age, from 21 t 10% at 1-3 days to 53 t 6% at 13-16 days (Table 5). Respunse to the thrumbuxane mimetic U-46619. In response to injection of 2 pg U-46619, all lambs at both ages showed an increase in pulmonary arterial pressure with almost no increase in left atria1 pressure. On days 1-3, pulmonary arterial and left atria1 pressures increased by 17 t 10 and 1 t 2 cmH,O, respectively; thus pulmonary vascular driving pressure increased by 16 t 10 cmH,O. On days 13-16, pulmonary arterial and left atria1 pressures increased by 11 t 8 and 1 t 1 cmH,O, respectively; again, pulmonary vascular driving pressure increased by

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10 -+ 9 cmH,O. The smaller increase in the older lambs was probably due to greater dilution of the fixed dose. Cardiac output. In five experiments (1 on days 1-3, 4 on days 13-16) cardiac output was measured after injection of a second dose of liposomes or Monastral blue. Cardiac output did not change after injection of either particle. DISCUSSION

Our preliminary histological survey shows that at birth lambs have very few identifiable intravascular macrophages, but by 2 wk macrophage numbers have increased -l-O-fold. This dramatic increase after birth has been documented in pigs in ultrastructural studies by Winkler and Cheville (29, 30), who rarely observed more than a few differentiated intravascular macrophages in nearterm or newborn piglet lungs but frequently observed fully differentiated mononuclear phagocytes, identified as pulmonary intravascular macrophages, in 7-day-old piglet lungs. It is likely that all of the Monastral blue, the particle we used to identify intravascular macrophages, was taken up by those cells. Cracker and associates (7,9) and Warner and colleagues (22, 27) examined particle clearance from the circulations of adult mammals with pulmonary intravascular macrophages (sheep, goats, calves, pigs). They found by ultrastructural analysis that the particles are almost exclusively contained in intravascular macrophages and are not located in endothelial cells or neutrophils. Albertine et al. (I) found similar results for Monastral blue in the adult goat. Because we used Monastral blue to positively identify a cell as a macrophage, it is possible there were macrophages in newborn lambs that were not identified because they did not take up Monastral blue. If so, we may have underestimated the number of macrophages in newborn lambs. However, our results still demonstrate a functional difference between the populations of cells in newborn and 2-wk-old lambs, because the cells in the newborn lambs did not phagocytize Monastral blue. Demonstrating Koch’s third postulate, there is an increase in pulmonary vascular reactivity in lambs to foreign particles from birth to 2 wk of age, and the increase is associated with a pulmonary vasoconstrictor that can be released by macrophages. The increase in pulmonary vascular reactivity is especially demonstrated by the in-

TABLE 3. Pulmonary vascular reactivity and thrumbuxane B, concentrations acrusspulmonary vascular bed in newburn lambs after injection of liposumes

All lambs Days

Paired subset Days l-3 Days

11

f-3

13-16

3t4

lkl

3t4

118+166

168t277

2t3 20*15*

Utl Q&l

2t2 20tl6*

564127 266+133

53t95 95t45

-50+141

5 3+33 171+103*

Values are group means t SD; n, no. of lambs. Liposomes (10 pmol) were injected intravenously. Driving pressures are pulmonary arterial minus left atria1 pressures. Net pulmonary thromboxane Bz production is syst,emic arterial minus pulmonary arterial concentrations. * Significantly different from paired subset, days 1-3 (P < 0.05). Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 10, 2019.

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to respond.

If

l-3

m

13-16

DAYS

OLD

DAYS

OLD

c

LUNG

LIVER

SPLEEN

The thromboxane mimetic U-46619 produced an increase in pulmonary vascular driving pressure in lambs on ckys l-3 as well as on days 13-l 6, thus demonstrating the ability of the newborn pulmonary circulation to vasoconstrict. Quinn et al. (16) also found that the pulmonary circulations of 3- to &day-old lambs vasoconstricted in response to U-46619. The fact that cardiac output fell or did not change after injection of particles confirms that the increase in pulmonary vascular driving pressure was due to an increase in resistance rather than an increase in flow. The pulmonary vascular reactivity and particle uptake in lambs on aays f 3-f 0 after injection of nposomes anu Monastral blue are similar to those found in other studies. Quinn et al. (16) compared the response of 3- to 5day-old lambs and of adult sheep to a bolus injection of propylene glycol. They found that pulmonary vascular resistance rose significantly less after injection in the lambs than it did in the older sheep. They attributed the difference in response to the absence of a significant population of pulmonary intravascular macrophages in the lambs. In 3-wk-old lambs, Teague et al. (20) found a dose-dependent increase in pulmonary arterial pressure wit.h Intralipid infusion and no change in left atria1 pressure or cardiac output. They also found a significant increase in plasma concentrations of thromboxane B, after infusion. Miyamoto et al. (14) injected 5.5 pmol radioactive liposomes into unanesthetized sheep and measured an increase in pulmonary arterial pressure of 31 cmH,O; the increase was blocked completely by indomethacin, an inhibitor of the cyclooxygenase cascade, and was blocked 75% by a specific thromboxane synthetase inhibitor. In addition, the lung retained 62% of the injected radioactivity. Albertine and Staub (2) injected anesthetized sheep with 1 ml/kg of 1% Monastral blue and measured an increase in pulmonary arterial pressure of 38 cmH,O; the increase was partly blocked by indomethacin. The increase in pulmonary vascular driving pressure with age appears to be mediated by an increase in thromboxane A, production within the pulmonary circulation, because simultaneous upstream and downstream sampling showed net production across the lungs. The sampling for thromboxane was done over several seconds at the peak of the change in pulmonary arterial pressure. This means that thromboxane was being produced substantially before sampling began. Because of the small blood volume of the lambs, we did not attempt to evaluate the kinetics of net thromboxane production; this ’

BLOOD

MARROW

FIG. 2. Uptake (as percent of injected dose) of intravenously injected ll’In-labeled liposomes by lung, liver, spleen, blood, and bone marrow of lambs l-3 and 13-16days old. Valuesare means t SD. Uptake by lung was significantly greater in older lambs, whereas uptake by liver was significantly greater in younger lambs (* P < 0.01).

crease in pulmonary vascular driving pressure with age after injection of a constant total dose (i.e., number of particles) of liposomes. The associated increase in net thromboxane production by the lung suggests that the increase in reactivity is mediated by thromboxane A,, which is released by intravascular macrophages in vitro (3). The increase in pulmonary vascular reactivity after injection in the older lambs is associated with an increase in the uptake of particles by macrophages in the pulmonary vascular bed. The greater pulmonary uptake of liposomes and Monastral blue in lambs 13-16 days old probably occurs because the population of mature pulmonary intravascular macrophages is increasing in number. The greater uptake of liposomes in the liver than in the lung of younger lambs shows that in newborn lambs, as in all mammals without pulmonary intravascular macrophages, the primary organ for particle clearance is the liver. With increasing age, the primary site of clearance shifts from liver to lung for particles injected systemically. The exception to this generalization is particles injected into the portal vascular bed, where liver macrophages (Kupffer cells) encounter them first and remove most of them (26,241. In 13- to 16-day-old lambs uptake is shared equally between liver and lung. If the lambs had further matured, they would have reached the higher uptake of liposomes in the lung measured by Miyamoto et al. (14) in adult sheep. The lack of response to particles in the younger lambs was not due to an inability of the pulmonary vasculature

1

1

1

dn

*/)

fil

l

l

10

1

11.#*

TABLE 4. Pulmonary vascular reactivity and thromboxane B, concentrations acrosspulmonary vascular bed in newborn lambs after injection of Monastrul blue pigment Changes in Pressures,

Days Days

1-3 13-16

Changes in Thromboxane

cmH,U

n

Pulmonary arterial

Left atria1

Driving pressure

6 5

3k2 25*15*

Otl 122

3+2 25&E*

Systemic arterial 68+101 768+638*

B, Concentrations,

pg/ml plasma

Pulmonary arterial

Net pulmonary production

80*79 3392264

-12273 429-t419*

Values are group means k SD; n, no. of lambs. Monastral blue (0.5 ml/kg of 1% solution) was injected intravenously. Driving pressures are pulmonary arterial minus left atria1 pressures. Net pulmonary thromboxane B2 production is systemic arterial minus pulmonary arterial concentrations. * Significantly different from days 1-3 (P < 0.05). Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 10, 2019.

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5. Mmastrul blue retention in lungs based on Monastrul blue concentrations in tissues n

Monastral Blue, mg/g Wet Lung

Lung Weight, g

Monastral Blue, % Injected

6 5

0.11t0.04 0*33+0.08*

94t29 138t37

21.2-t-9.8 53.4t6.1”

Values are group means k SD; n, no. of lambs. * Significantly ent from days 1-3 (P < 0.01).

LAMBS

quests: School of Veterinary Medicine, Dept. of Anatomy and Cell Biology, University of California, Davis, CA 95616.

TABLE

Days 1-3 Days 13-16

IN NEWBORN

Received 10 January 1992; accepted in final form 1 July 1992. REFERENCES

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probably accounts for the large SD associated with the absolute thromboxane concentrations in Tables 3 and 4. However, we are concerned with the difference across the lung, which was always positive. The source of the thromboxane may be the intravascular macrophages themselves. Cells that are putatively macrophages do produce thromboxane in culture (3, 4). However, Miyamoto (13) found that the pulmonary hemodynamic response and thromboxane production after liposome infusion in sheep were attenuated if a plateletactivating factor antagonist was infused before the liposomes. Thus the macrophages may be stimulated to release platelet-activating factor, which would then stimulate other pulmonary cells or the macrophages themselves to release thromboxane (19). The positive change in thromboxane concentration in the pulmonary artery after injection of both particles indicates a possible systemic source. In the older lambs, in which net pulmonary production was positive, thromboxane could have been recirculating from the lung. In the younger lambs, where pulmonary production was negative or zero, there must have been a systemic source. Liver macrophages also produce eicosanoids when stimulated by foreign particle uptake. However, not much thromboxane appears in hepatic venous blood because the hepatocytes rapidly take up and metabolize it; it has been reported that, rather than thromboxane, the primary eicosanoid from the liver macrophages is prostaglandin D, (21). In conclusion, this study fulfills Koch’s third postulate as applied to the pulmonary intravascular macrophages in sheep; in the absence of mature macrophages the pulmonary response to foreign particles (increase in driving pressure, thromboxane production, phagocytosis) is greatly attenuated. With the appearance (or maturity) of these cells, the pulmonary response appears. Although experiments in adult sheep demonstrating inhibition of macrophages will confirm these findings, this natural experiment effectively links pulmonary intravascular macrophages to the pulmonary vascular reactivity that can initiate a cascade of processes leading to lung injury, disease, and death. We thank Dr. Kurt H. Albertine, Elizabeth Schultz, and Todd Bloom for expert assistance, and Upjohn (Kalamazoo, MI) for donation of U-46619. This study was supported by National Heart, Lung, and Blood Institute Grants HL-25816 (Program Project) and HL-44122 (First Award to J. Y. Westcott) and a National Institutes of Health National Research Service Award to K. E. Longworth. Present address of K. E. Longworth and address for reprint re-

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