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J Appl Physiol 81: 1730-1738, 1996;
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Journal of Applied Physiology
Vol. 81, No. 4, pp. 1730-1738, October 1996
PULMONARY CIRCULATION AND LUNG FLUID BALANCE

Chronic interleukin-2 treatment in awake sheep causes minimal or no injury to the lung microvascular barrier

E. Heidi Jerome, Keiji Enzan, Dominique Douguet, Dachuan Lei, Gary Jesmok, Carol W. Johnson, Maritza Neuburger, and Norman C. Staub

Departments of Anesthesia, Medicine, Physiology, and Cardiovascular Research Institute, University of California, San Francisco, California 94143-0542; and Cetus Corporation, Emeryville, California 94608

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Jerome, E. Heidi, Keiji Enzan, Dominique Douguet, Dachuan Lei, Gary Jesmok, Carol W. Johnson, Maritza Neuburger, and Norman C. Staub. Chronic interleukin-2 treatment in awake sheep causes minimal or no injury to the lung microvascular barrier. J. Appl. Physiol. 81(4): 1730-1738, 1996.---Interleukin-2 (IL-2) is reputed to cause a "vascular leak syndrome." We studied pulmonary hemodynamics and lymph dynamics in six sheep treated for 7 days with IL-2 (1.8 million IU/kg twice daily or 1.8 million IU/kg each day as a continuous infusion). Lung lymph flow increased from 4.8 ± 2 ml/15 min pre-IL-2 to 14.4 ± 6.8 ml/15 min on the seventh day of IL-2. The lymph-to-plasma protein concentration ratio was unchanged (0.70 ± 0.06 vs. 0.63 ± 0.13). The plasma-to-lymph equilibration half-time of radiolabeled albumin was 2.0 ± 0.6 h pre-IL-2 and 1.0 ± 0.7 h on day 7 of IL-2. Pulmonary arterial pressure was 24 ± 7 cmH2O pre-IL-2, increased to 32 ± 4 cmH2O on the fourth day of IL-2, and returned to 29 ± 5 cmH2O on the seventh day of IL-2. Extravascular lung water was normal (4.07 ± 0.25 g/g dry lung). To clearly determine whether the increase in lung lymph flow was due to hemodynamic changes or to increased leakiness of the microvascular barrier, we volume loaded six sheep with lactated Ringer solution before and after 3 days of IL-2 treatment (1.8 million IU/kg twice daily). Lung lymph flows increased fivefold during 4 h of crystalloid infusion compared with baseline and were higher after 3 days of IL-2. However, lymph-to-plasma protein concentration ratios decreased to the same low levels pre- and post-IL-2 (0.39 ± 0.06 vs. 0.41 ± 0.10), indicating an intact microvascular barrier. Extravascular lung water was elevated (5.56 ± 0.39 g/g dry lung) but was not different from lung water in three volume-loaded control sheep (4.87 ± 0.53 g/g dry lung). We conclude that IL-2 causes minimal or no injury to the pulmonary microvascular barrier and that volume expansion during IL-2 treatment can cause hydrostatic pulmonary edema.

pulmonary edema; pulmonary circulation; lung injury

INTRODUCTION

RECOMBINANT INTERLEUKIN-2 (IL-2) is used to treat selected patients with advanced cancers. However, treatment-related systemic and pulmonary toxicity often limits the amount of IL-2 a patient can receive (19, 27). Systemic effects include hypotension, oliguria, ascites, and weight gain. Pulmonary effects range from mild dyspnea to severe respiratory distress requiring ventilatory support. Pleural effusions are also reported in some patients (17, 27, 30).

Most investigators attribute the systemic and pulmonary edema to a "vascular leak syndrome." Studies in rats and mice (8, 9, 23, 29) have shown increased pulmonary microvascular leakiness, although Ferro et al. (10) identified an increase in pulmonary hydrostatic pressure as the etiology of pulmonary edema in guinea pig lungs. In human trials of IL-2, investigators have usually attributed hypotension, oliguria, and edema to systemic and pulmonary vascular leakiness based on the data from small-animal studies. However, several investigators (2, 17, 20, 22) have identified vasodilatation, myocardial dysfunction, and hypoproteinemia in patients undergoing IL-2 therapy. These hemodynamic effects may account, in part, for the systemic and pulmonary edema and the hypotension that accompany IL-2 treatment.

Our original intent when we undertook this study was to determine the mechanism by which IL-2 caused pulmonary microvascular injury. We studied awake sheep treated with IL-2 regimens similar to those used in patient protocols. We investigated pulmonary hemo- and lymph dynamics, systemic hemodynamics, and fluid balance as well as general symptoms during 7 days of IL-2 treatment. However, we were surprised to find that, although lung (caudal mediastinal node) lymph flow increased and the sheep appeared ill with lethargy, anorexia, diarrhea, and fever, we could not demonstrate increased pulmonary or systemic vascular leakiness. We, therefore, undertook additional studies to determine whether we could induce pulmonary edema by volume expansion during IL-2 treatment. These sheep developed hydrostatic, not increased leakiness, pulmonary edema.

METHODS

Preparation

We obtained institutional consent from our Committee on Animal Research to conduct these studies on IL-2. We placed chronic indwelling catheters in 15 sheep (24-39 kg) of mixed breeds at two sterile surgeries. For each operation, the sheep were anesthetized with thiopental sodium (15 mg/kg iv), endotracheally intubated, and maintained on halothane (1% in 50% oxygen) via a positive-pressure ventilator. For postoperative analgesia, bupivacaine (3 ml of 0.25%) intercostal nerve blocks were placed at several interspaces above and below the thoracotomy incisions before the sheep awoke from anesthesia. The sheep received prophylactic penicillin (20,000 U/kg) and streptomycin (25 mg/kg) twice daily for several days postoperatively.

At the first surgery, through a left thoracotomy at the ninth intercostal space, we cauterized the pleura over the lower esophagus and left hemidiaphragm and cut the caudal mediastinal node (CMN) to remove nonpulmonary lymph flow to the node (26). Through a left thoracotomy at the fourth intercostal space, we placed a 3.5F thermistor (Edwards Laboratories, Santa Ana, CA) and a polyvinyl catheter in the main pulmonary artery and placed another polyvinyl catheter in the left atrium. The sheep recovered from surgery for 5-7 days before the second operation was performed.

At the second surgery, through a right thoracotomy at the seventh intercostal space, we cauterized the pleura over the lower esophagus and the right hemidiaphragm. We cannulated the efferent duct of the CMN (32) with polyvinyl tubing (Tygon, 0.03 in. ID; Norton Plastics, Akron, OH) that had been coated with tridodecylmethylammonium chloride-heparin (Polysciences, Warrington, PA). Through a right neck incision, we also placed polyvinyl catheters in the right atrium and thoracic aorta via the jugular vein and carotid artery, respectively. We allowed the sheep to recover until the lymph was clear and flowed at a stable rate. We flushed the catheters every other day with heparin (1,000 U/ml). The sheep remained in mobile cages where they had free access to food and water after surgery and during the experiments. The sheep wore instrumentation vests to protect indwelling catheters.

Experimental Protocols

Chronic IL-2 treatment. Four sheep received IL-2 (1.8 million IU/kg iv) in 50 ml of 5% dextrose over 30 min twice daily for 7 days. Two sheep received continuous infusions of IL-2 (1.8 million IU/kg iv) in 50 ml of 5% dextrose over 24 h for 7 days. The IL-2 was continuously infused by a portable pump (Infumed 2000, Medfusion Systems, Norcross, GA) contained within the sheep's vest. The bolus dose of IL-2 was chosen based on preliminary experiments in which we found signs similar to those of patients during IL-2 treatment, and the sheep survived for 7 days at this dose. The continuous dose, which is one-half of the bolus dose over the entire study, was chosen based on preliminary experiments in which we found greater systemic toxicity from continuous compared with bolus infusion. The doses of IL-2 used are one to two times those used in humans. Both the bolus and continuous infusion regimens have been used to treat patients.

Sheep were continuously monitored during 10-h studies before receiving IL-2 and on the first, fourth, and seventh days of IL-2 treatment. We weighed the sheep before IL-2 treatment and on the fourth and seventh days of IL-2. We measured intake (food, water, and infusions) and output (urine, stool, blood, and lung lymph) daily. During the experiments, we measured aortic pressure, pulmonary arterial pressure, and left atrial pressure with lightweight continuously flushing transducers (Medex, Hilliard, OH) attached to the sheep's vest. All pressures were referred to the base of the lung and were continuously recorded on a multichannel recorder (model 7, Grass Instrument, Quincy, MA). We determined cardiac output by thermodilution on a computer (model 3500, Mansfield Electronics, Mansfield, MA) and calculated systemic and pulmonary vascular resistances every 30-60 min. We measured blood temperature continuously and counted respiratory rate and heart rate hourly. We collected lung lymph in heparinized graduated tubes and measured the volume of lymph hourly.

We measured lymph and plasma protein concentrations with the biuret method and albumin with the bromcresol green method in an automated device (model AAII, Technicon Instruments, Tarrytown, NY). We calculated the lung lymph-to-plasma total protein concentration ratio (Clymph/Cp). We determined pH, arterial PCO2 (PaCO2), arterial PO2 (PaO2) (model 158, Corning Medical Products, Medfield, MA), and hematocrit hourly. If the hematocrit increased 4% or more over the pre-IL-2 value, we slowly infused 1 liter of lactated Ringer solution intravenously at the end of a 10-h study or at any time on nonstudy days to prevent dehydration.

Hourly leukocyte concentrations in blood and lymph were determined in an automated device (model F, Coulter Electronics, Hialeah, FL). On the first and seventh days of IL-2, a 100-cell differential count was made after smears of blood and lymph were stained by a modified Wright stain.

PLASMA ESCAPE AND PLASMA-TO-LYMPH TRACER PROTEIN EQUILIBRATION RATES. In five sheep, we injected 5-15 µCi of 125I- or 131I-labeled human serum albumin (Merck Frosst Canada, Kirkland, Quebec) intravenously on the day before IL-2 treatment and on the fourth and seventh days of IL-2. The radioactive tracer was given 2 h after the morning dose of IL-2 in the sheep treated with bolus IL-2 and after a 2-h stable baseline in the sheep receiving the continuous infusion of IL-2. We increased the dose of tracer or gave a different isotope in successive experiments. After the injection of radioactive tracer, we collected lung lymph and plasma every 15 min for 4 h then every 30 min for 3 h. We determined the specific activity (concentration of albumin-bound radioactivity per gram of albumin) in plasma and lymph by counting 125I or 131I radioactivity in a gamma counter (Autogamma 3002, Packard Instrument, Downers Grove, IL) and dividing by the albumin concentration in plasma or lymph. We corrected for successive doses of the same isotope and for crossover of 131I to 125I activity by subtracting the concentration of radioactivity in the baseline plasma or lymph samples (before isotope injection) from all samples collected on that day.

The plasma escape and plasma-to-lymph equilibration rates were obtained by plotting the specific activity of albumin in plasma and the difference between the plasma and lymph specific activities over the time course of the experiment on a logarithmic scale. The results for one experiment are shown in Fig. 1. The plasma specific activity and the plasma-to-lymph difference in specific activity decreased monoexponentially (straight line on semilog plot). We determined the straight line of best fit over the time course of the experiment by least squares regression analysis and from that calculated the half-time of the process.
Fig. 1. Specific activity of radiolabeled albumin in plasma and lung lymph and difference between plasma and lymph (Plasma-lymph) values in 1 sheep (S-82) on days 0 (A) and 7 (B). cpm, Counts/min. Solid lines, plasma escape rate and plasma-to-lymph equilibration rates from which half-times were determined. Plasma escape rate was unchanged, whereas plasma-to-lymph equilibration rate was faster after 7 days of interleukin-2 (IL-2) treatment.
[View Larger Version of this Image (15K GIF file)]

PLASMA VOLUME CALCULATIONS. We multiplied the concentration of 125I or 131I radioactivity in the injectate by the volume of the injectate and divided this result by the concentration of 125I or 131I radioactivity in a sample of plasma taken 5 min after the tracer injection to determine plasma volume.

We determined the percentages of free iodine in plasma, lymph, and radioactive-albumin injectates by trichloroacetic acid precipitation in four sheep and found them to be 1.8 ± 0.9, 2.3 ± 0.8, and 3.2 ± 1.1%, respectively. Radioactive iodine uptake in thyroid tissue at postmortem was 3.2 ± 1.2% of that injected. These low levels of free iodine indicate that most radioactivity remained bound to albumin during the equilibration studies.

IL-2 treatment and volume loading. Six additional sheep were volume loaded before and after 3 days of bolus IL-2 treatment. During the first experiment in each sheep, after a 3-h stable baseline, we infused warmed lactated Ringer solution intravenously to raise the left atrial pressure 10-15 cmH2O and to maintain it at that level for 4 h [range 5.0-10.6 liters; 8.3 ± 2.6 (SD) liters]. These criteria, which resulted in a large infused volume of Ringer lactate, were chosen to determine whether pulmonary edema was caused by a hydrostatic pressure increase or an increased leakiness of the microvascular barrier. During the experiment, we measured aortic pressure, left atrial pressure, pulmonary arterial pressure, cardiac output, lung lymph flow, and plasma and lymph protein and albumin concentrations every 15 min and pH, PaCO2, and PaO2 hourly.

After the sheep had recovered from the first volume-loading experiment, we treated them with IL-2. Each sheep received 1.8 million IU/kg twice daily for a total of six doses. We chose this regimen because bolus injection and continuous infusion gave similar results in the earlier studies and because the sheep in the earlier studies developed hemodynamic and lymph-dynamic responses to IL-2 by 3 days without severe systemic toxicity.

Three days after the first experiment, we conducted a second volume-loading experiment. The final dose of IL-2 was given just before the second volume-loading experiment started. We then measured the same variables as in the previous experiment for a 3-h stable baseline, followed by a 4-h infusion of lactated Ringer solution intravenously at the same rate and volume as in the previous experiment. We collected lymph and plasma samples and determined protein, albumin, hematocrit, and blood gas tensions as in the previous experiment. The sheep were weighed before each volume-loading experiment.

To compare the effects of volume loading and IL-2 treatment with volume loading alone on extravascular lung water measurements, three additional sheep were not given IL-2 but underwent two volume-loading experiments 3 days apart.

At the end of the second volume-loading experiment and after the final experiment in the sheep treated with IL-2 for 7 days, we anesthetized the sheep with pentobarbital sodium (60 g iv) and removed the lungs for histology and lung water determination. Additional tissues (described in Pathology and histology) were also removed for histology.

Postmortem Studies

Extravascular lung water measurements. The lungs, except for the left lower lobes that were fixed for histological study, were weighed and homogenized in a blender with an equal amount of water. Aliquots were centrifuged at 1,600 g for 1 h, and the resulting supernatant was analyzed for hemoglobin concentration. Determination of hemoglobin concentration was also performed on a final arterial blood sample. The aliquots of lung tissue and supernatant were placed in an oven and dried to a stable weight. The formula of Selinger et al. (31) was used to determine extravascular lung water.

Pathology and histology. At postmortem, we grossly examined the lungs, lymph nodes, and abdominal organs. We removed the lungs from the thorax and filled the left lower lobe airway with 10% Formalin without overdistending the airways. We then cut the left lower lobe away from the rest of the lung and immersed it in Formalin for 72 h. After the volume-loading experiments, we also removed the heart, kidneys, spleen, CMN, mesenteric lymph node, inguinal lymph node, and pieces of the liver, stomach (abomasum), and ileum and fixed these in Formalin. We trimmed, dehydrated, and embedded the tissues in paraffin. We cut sections, mounted them on slides, and stained them with hematoxylin and eosin. One of us (C. W. Johnson), a veterinary pathologist, in a blinded fashion, examined the lungs from two sheep after 7 days of continuous IL-2 infusion and the lungs and abdominal organs from six IL-2-treated volume-loaded sheep and from three sheep that were only volume loaded. The tissues were examined for edema and cellular infiltrates.

Recombinant IL-2

The IL-2 was human recombinant material, produced and supplied by Cetus (Emeryville, CA) in premixed sterile vials containing 18 million IU of lyophilized powder that was reconstituted in sterile water, then diluted in 50 ml of 5% dextrose in water. Lots of IL-2 were tested by Cetus and by us with a Limulus amoebocyte lysate assay and were found to contain <0.04 ng of endotoxin/18 million IU of IL-2.

Statistics

We determined that each value for all variables from the sheep treated with continuous-infusion IL-2 fell within the tolerance limits for the same variables of sheep treated with bolus IL-2, so we grouped these data.

We expressed all data as group means ± SD except for the morphological studies. In the sheep treated with IL-2 for 7 days, we compared data from baseline and at 6-7 h after IL-2 on days 1, 4, and 7 by repeated-measures analysis of variance (ANOVA), followed by the Newman-Keuls test when significant differences were found. The weight, maximum temperature, plasma volume, plasma escape, and plasma-to-lymph equilibration values before IL-2 and on days 4 and 7 of IL-2 treatment were also compared by ANOVA, followed by the Newman-Keuls test.

In the sheep treated with IL-2 plus volume loading, we compared data from baseline conditions and during the fourth hour of volume loading by repeated-measures ANOVA, followed by the Newman-Keuls test. Extravascular lung water values from the three sheep treated only with volume loading and from the six sheep treated with IL-2 plus volume loading were compared by unpaired Student's t-test. We accepted P < 0.05 as indicating a significant difference in all comparisons.

RESULTS

Chronic IL-2 Treatment

General. Most sheep became less active on the first day of IL-2 treatment and all sheep were listless by the third day. Compared with normal pre-IL-2 temperatures of 39.3 ± 2°C, the sheep developed maximum temperatures of 40.4 ± 0.1°C (P < 0.05) on each day they received IL-2, and those receiving bolus IL-2 developed shaking chills 30 min after the bolus was given. All sheep stopped eating and drank little after 1-2 days of IL-2 treatment. They developed soft stools by 3 days of treatment and watery diarrhea by 5 days of treatment.

Weight had decreased by 10% on day 4 of IL-2 compared with pre-IL-2 and by an additional 5% by day 7 of IL-2 (Table 1) despite a measured positive fluid balance of 4.4 ± 1.5 liters over 7 days of IL-2. Plasma volume tended to decrease by 9% on day 4 and by 4% on day 7 compared with pre-IL-2 values but was not statistically significant. Plasma albumin concentration decreased from 2.7 ± 0.2 g/dl pre-IL-2 to 2.3 ± 0.3 g/dl on the seventh day of IL-2. A mild metabolic acidosis developed by day 7. Because of a 14% increase in hematocrit on day 4 compared with pre-IL-2, five of the six sheep received 2 ± 1 liters of Ringer lactate over the final 3 days of the study. Despite these indications of intravascular volume depletion, the plasma escape rate was unchanged over 7 days of IL-2 (Table 1).

Table 1. Markers of volume status during chronic IL-2 treatment

Day 0  Day 4  Day 7 
Weight, kg 38.6 ± 6.0  34.9 ± 7.0* 32.8 ± 7.3*
Hematocrit, %  31 ± 4  36 ± 4* 35 ± 4 
Plasma volume, liters 1.9 ± 0.4  1.7 ± 0.2  1.8 ± 0.4 
Plasma escape rate (t1/2), h 9.7 ± 2.8  10.2 ± 3.5  9.8 ± 1.5
Values are means ± SD. IL-2, interleukin-2; t1/2, half-time. * Significant difference from day 0 by analysis of variance (ANOVA) and Newman-Keuls test, P < 0.05.

Systemic hemodynamics. All sheep developed systemic hypotension in response to the initial dose of IL-2, and systemic pressure remained low on days 4 and 7 of IL-2 (Table 2). Left atrial pressure also decreased as did cardiac output. Heart rate increased by hours 6-7 each day that IL-2 was given (138-146 beats/min) but returned to baseline each morning (107-120 beats/min). Systemic vascular resistance was unchanged throughout the experiment.

Table 2. Hemodynamics and lymph dynamics during IL-2 treatment

Day 1 
 Day 4 
 Day 7 
Baseline Hours 6-7 Baseline Hours 6-7 Baseline Hours 6-7
Aortic pressure, mmHg 83 ± 6  79 ± 6  72 ± 8  70 ± 12* 60 ± 14* 60 ± 14*
Pulmonary arterial pressure, cmH2O 24 ± 7  26 ± 3  25 ± 3  32 ± 4*Dagger 24 ± 4  29 ± 5 
Left atrial pressure, cmH2O 7 ± 4  5 ± 3  6 ± 4  3 ± 4  5 ± 4  1 ± 3*dagger Dagger
Cardiac output, l/min 6.6 ± 0.9  7.1 ± 1.4  5.0 ± 1.1* 5.0 ± 1.3* 4.1 ± 0.6* 4.1 ± 0.8*
Lung lymph flow, ml/h 4.8 ± 2  10.5 ± 6.1*dagger 4.6 ± 1.3  8.9 ± 4.3  8.5 ± 3.4  14.4 ± 6.8*dagger Dagger
Lymph-to-plasma protein concentration ratio 0.70 ± 0.06  0.72 ± 0.03  0.76 ± 0.05  0.74 ± 0.07  0.63 ± 0.14dagger 0.63 ± 0.13dagger
Values are means ± SD. All pressures are referred to base of lung. Significant difference (P < 0.05) by ANOVA and Newman-Keuls test from: * day 1 baseline; dagger day 4 baseline; Dagger day 7 baseline.

Pulmonary effects of IL-2. Pulmonary arterial pressure was increased at hours 6-7 on day 4 of IL-2 compared with day 1 baseline (pre-IL-2) and day 7 baseline (Table 2). Pulmonary vascular resistance was increased at hours 6-7 on day 4 (6.3 ± 2.6 cmH2O · l-1 · min) and day 7 (7.1 ± 2.8 cmH2O · l-1 · min) compared with the day 1 baseline value (2.4 ± 0.8 cmH2O · l-1 · min). Lung lymph flow increased two- to threefold in response to IL-2 on days 1, 4, and 7 (Fig. 2, Table 2). The Clymph/Cp was unchanged on day 4 compared with day 1 but decreased by day 7 (Fig. 2, Table 2).

Fig. 2. Lung lymph flow and plasma-to-lymph protein concentration ratio in 6 sheep on days 1, 4, and 7 of IL-2 treatment. Data are means. Lung lymph flow increased and lymph-to-plasma protein concentration ratio decreased in response to IL-2.
[View Larger Version of this Image (17K GIF file)]

The plasma-to-lymph equilibration rate was 2.0 ± 0.6 h pre-IL-2, 1.3 ± 0.6 h on day 4, and 1.0 ± 0.7 h on day 7. The day 7 value is significantly shorter than the pre-IL-2 value.

PaO2 decreased from 86 ± 8 Torr pre-IL-2 to 76 ± 8 and 77 ± 9 Torr at hours 6-7 on days 4 and 7, respectively. PaCO2 decreased from 37 ± 5 Torr pre-IL-2 to 32 ± 6 and 33 ± 4 Torr at hours 6-7 on days 4 and 7, respectively. Only the day 4 value reached significance. The changes in PaCO2 may be partial compensation for the small but real decrease in pH from 7.5 ± 0 on day 1 to 7.4 ± 0.1 on day 7. Respiratory rate decreased on days 4 (27 ± 7 breaths/min) and 7 (24 ± 5 breaths/min) at baseline compared with the day 1 (67 ± 36 breaths/min) value.

Leukocyte counts. The number of circulating leukocytes doubled by day 7 compared with days 1 and 4 (Table 3). The percent lymphocytes in blood decreased after the first dose of IL-2 on day 1, then increased by day 7. Baseline leukocyte counts in lung lymph (99% lymphocytes) were unchanged during baseline on days 1, 4, and 7 but decreased after administration of IL-2 on each of these days. However, the lymph leukocyte traffic (leukocyte count in lymph × lung lymph flow) nearly doubled by day 7 compared with days 1 and 4.

Table 3. Blood and lymph leukocyte counts during chronic IL-2 treatment

Day 1 
 Day 4 
 Day 7 
Baseline Hours 6-7 Baseline Hours 6-7 Baseline Hours 6-7
Blood leukocytes
  Total, 103/µl 8 ± 3  10 ± 3  10 ± 3  8 ± 3* 20 ± 10  17 ± 9 
  Lymphocytes, %  54 ± 14  24 ± 13dagger 64 ± 16dagger 58 ± 22 
Lymph leukocytes, 103/µl 43 ± 13  12 ± 16  44 ± 19  22 ± 14  48 ± 21  21 ± 13Dagger
Lymph leukocyte flow, 103/µl × ml/15 min 195 ± 67  69 ± 45dagger 185 ± 135  169 ± 115  363 ± 138*§ 271 ± 153
Values are means ± SD. Significant difference (P < 0.05) by ANOVA and Newman-Keuls test from: * day 1 baseline and day 4 hours 6-7; dagger every other value in row; Dagger day 1 hours 6-7; § day 4 baseline.

Postmortem examination. The lungs appeared grossly normal without evidence of edema except in one sheep that had pneumonia involving part of the right upper and lower lobes. Microscopic examination confirmed a focal interstitial infiltrate in this sheep, whereas the remainder of this lung and a second sheep's lungs were normal (Fig. 3). Gravimetric extravascular lung water in all six sheep was normal at 4.07 ± 0.25 g/g dry lung.


Fig. 3. IL-2-treated sheep lung (A) showing bronchiole, vessel, and surrounding alveoli is not different from normal sheep lung (B) showing similar structures. Lung from a sheep receiving intravascular fluids (C) shows interstitial edema (thick arrow) surrounding a bronchiole without edema of alveolar wall (thin arrow). Lung from a sheep treated with IL-2 and intravascular fluids (D) shows similar interstitial edema (thick arrow) without alveolar wall edema (thin arrow). A dilated lymphatic (open arrow) and small vessel are also present. Magnification, ×250.
[View Larger Versions of these Images (91 + 74 + 97 + 105K GIF file)]

The stomach, abomasum, and intestine were empty, which is unusual in healthy sheep, but showed no evidence of edema. The CMN that drains lung lymph was enlarged.

IL-2 Treatment and Volume Loading

Systemic effects. In the six sheep treated with volume loading before and after IL-2, aortic pressure increased during volume loading both pre- and post-IL-2 compared with baseline values but was lower during baseline and volume infusion after IL-2 compared with pre-IL-2 values (Table 4). Pulmonary arterial and left atrial pressures increased during volume loading (the increase in left atrial pressure pre-IL-2 was the controlled variable) before and after IL-2. Cardiac output increased during volume loading after IL-2 compared with both pre-IL-2 and post-IL-2 baselines. The sheep lost 5.2 ± 3% of body weight over 3 days. Plasma albumin decreased from 2.8 ± 0.1 g/dl during pre-IL-2 baseline to 2.5 ± 0.2 g/dl during the post-IL-2 baseline.

Table 4. Hemodynamics and lymph dynamics during IL-2 treatment and volume loading

Pre-IL-2
Post-IL-2
Baseline Volume Baseline Volume
Aortic pressure, mmHg 83 ± 7  105 ± 12* 71 ± 7dagger 82 ± 16*dagger
Pulmonary arterial pressure, cmH2O 23 ± 3  34 ± 4* 24 ± 3  38 ± 11*
Left atrial pressure, cmH2O 3 ± 5  14 ± 6*  -2 ± 2  13 ± 3*
Cardiac output, l/min 5.0 ± 1.0  6.0 ± 1.6* 5.4 ± 1.2  7.4 ± 2.5*
Lung lymph flow, ml/h 7 ± 4  36 ± 17* 13 ± 9  72 ± 23*dagger
Lymph-to-plasma protein concentration ratio 0.66 ± 0.05  0.38 ± 0.05* 0.73 ± 0.05  0.41 ± 0.10*
Values are means ± SD. Significant difference (P < 0.05) by ANOVA and Newman-Keuls test from: * same day baseline; dagger pre-IL-2 value at same time.

Pulmonary effects. Lung lymph flow increased fivefold during volume loading before and after IL-2 (Fig. 4, Table 4). Lymph flow was greater during volume loading post-IL-2 than pre-IL-2, but baseline values were not different. Although lung lymph flow increased, the Clymph/Cp decreased both pre- and post-IL-2 in a parallel fashion and were not different (Fig. 4, Table 4). The Clymph/Cp decreased to the same low value of ~0.40 at the end of volume loading.

Fig. 4. Lung lymph flow and lymph-to-plasma protein concentration ratio in 6 sheep during volume loading before and after 3 days of IL-2. Data are means ± SD. Lung lymph flow increased more after IL-2 treatment compared with pre-IL-2 studies, whereas lymph-to-plasma protein concentration ratio decreased to the same low levels both before and after IL-2, indicating an intact microcvascular barrier.
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PaO2 decreased from 88 ± 8 Torr during the pre-IL-2 baseline to 72 ± 7 Torr during volume loading after IL-2. PaO2 during volume loading pre-IL-2 was 81 ± 12 Torr, not different from baseline. PaCO2 and pH were unchanged.

Postmortem examination. Grossly, the lungs appeared normal. On microscopic examination, moderate interstitial edema liquid was located in the peribronchovascular cuffs but not in the alveoli. The control sheep that were volume loaded but not treated with IL-2 showed a similar pattern of interstitial edema (Fig. 3). Gravimetric extravascular lung water was increased in the IL-2-treated sheep that were also volume loaded (5.56 ± 0.39 g/g dry lung) but was not different from lung water in the control volume-loaded sheep (4.87 ± 0.53 g/g dry lung). Multiple small areas of interstitial pneumonia were noted in several of the IL-2 treated and control sheep. This is a common finding in field sheep (15).

The stomach, abomasum, and intestine were empty in the IL-2-treated sheep. Interstitial edema was present in the small intestine and in mesenteric and inguinal nodes and CMNs of the IL-2-treated and control sheep. Lymphoreticular hyperplasia was present in the lymph nodes of the IL-2-treated sheep. Eosinophilic infiltrates were present in the intestine of IL-2-treated sheep. Kupffer cell hyperplasia and periportal pleocellular infiltrates were present in the livers of IL-2-treated sheep. Other abdominal organs showed no differences between IL-2-treated and control sheep.

DISCUSSION

The sheep treated with IL-2 for 7 days did not develop pulmonary edema either histologically or by lung water measurements. In contrast, the sheep treated with IL-2 for 3 days and volume loaded did develop interstitial pulmonary edema histologically and increased lung water. The amount of edema formed was similar to that in volume-loaded sheep that were not treated with IL-2.

Lung lymph flow increased in response to IL-2 in both the sheep treated for 7 days and the sheep treated for 3 days and then volume loaded. Is this increase in lymph flow in both groups of sheep and the interstitial edema in the volume-loaded IL-2 treated sheep due to increased microvascular leakiness or to pressure alterations in the pulmonary vasculature?

The sheep treated with IL-2 for 7 days showed small increases in pulmonary arterial pressure (on day 4) and pulmonary vascular resistance (on days 4 and 7). The volume-loaded and IL-2-treated sheep did not show this increase in pulmonary arterial pressure or pulmonary vascular resistance compared with the control volume-loaded sheep, perhaps because the large changes in pulmonary hemodynamics in response to volume loading masked more subtle changes due to IL-2. Plasma protein osmotic pressure decreased after IL-2 treatment. Both the increase in hydrostatic pressure and resistance and the decrease in osmotic pressure would alter the Starling equation in favor of increased liquid filtration across an intact microvascular barrier (3) during IL-2 treatment.

Two additional explanations for an increase in lung lymph flow in the absence of increased microvascular leakiness are that 1) an increased pulmonary microvascular surface area could account for an increase in lung lymph flow in the absence of a rise in pulmonary arterial pressure, analogous to the effects of mild to moderate exercise in sheep (21). Stuntz and colleagues found evidence for an increase in the surface area of the intestinal vasculature in response to IL-2 in mice (34) and dogs (33) in the absence of injury; and 2) IL-2 might directly affect the CMN and lymphocytes rather than the lungs. Lymph nodes can alter efferent lymph flow and protein concentration, particularly in response to lymphocyte traffic (1, 25). The CMN may filter more lymph and protein from blood in response to increased lymphocyte flow during IL-2 treatment. We have no evidence that this did or did not occur.

Of course, lung microvascular injury may also have caused the increase in lymph flow during IL-2 treatment. Such injury would have to be minimal to account for the decrease in the Clymph/Cp that we observed during the rise in lymph flow.

To determine whether altered pressure or increased leakiness accounted for the increase in lung lymph flow, we measured the plasma-to-lymph equilibration half-time. The faster (45%) equilibration half-time on days 4 and 7 of IL-2 compared with pre-IL-2 lies midway between the faster (25%) equilibration rate that occurs with increased hydrostatic pressure and the much faster (75%) rate that occurs with increased leakiness of the lung vasculature (35).

Because the evidence for hemodynamic changes vs. increased leakiness as the cause of increased lung lymph flow was still equivocal, we proceeded with the volume-loading experiments. The increase in lymph flow caused by volume loading was accompanied by a decrease in the Clymph/Cp. The very low level to which Clymph/Cp fell during volume infusion even after IL-2 is strong evidence that the barrier function of the pulmonary vascular endothelium was essentially normal. In the presence of microvascular injury, the plasma proteins would have crossed the endothelial barrier into the lymph and the Clymph/Cp would have remained high.

Focal infiltrates were present histologically in the lungs of IL-2-treated and control sheep. This is a frequent finding in sheep raised in an outdoor environment. It is unlikely these small areas of lung inflammation confounded our results because they were present in control as well as in IL-2 treated sheep. Additionally, we found no evidence of significant endothelial injury that one might expect in response to inflammation.

Several investigators who studied the pulmonary effects of IL-2 concluded that IL-2 causes increased vascular leakiness in the lungs. Downie et al. (5), however, found only a small increase in cultured bovine endothelial cell permeability even at very high doses of IL-2. The experiments done in mice (7, 11, 23, 24, 28) and in rats (9) used doses that were 10-20 times larger than the doses used in our sheep or in humans. The massive doses of IL-2 or a species difference between the rodents' and sheep's response to IL-2 may account for these quite different effects. Even with very high doses of IL-2, Ferro et al. (10) found that increased hydrostatic pressure was the most likely cause of pulmonary edema in perfused guinea pig lungs.

Glauser et al. (13) and Duke et al. (6) conducted studies in sheep treated chronically with an IL-2 regimen similar to ours. Our hemodynamic and lymph-dynamic data are similar to theirs. The key points are a two- to fourfold increase in CMN efferent lymph flow, whereas Clymph/Cp was unchanged in response to IL-2. They also found a moderate increase in pulmonary arterial pressure. Glauser et al. (13) reported increased gravimetric lung water in IL-2-treated sheep, but his sheep received 3 liters of saline over 3 days, which is more than we gave our chronically IL-2-treated sheep over 7 days. When we volume loaded sheep, we, too, found a 20-30% increase in lung water. In additional studies in anesthetized sheep with left atrial pressure elevation, Glauser et al. found no difference in the protein reflection coefficient in IL-2-treated vs. untreated animals, which is exactly what we found.

On the other hand, Klausner et al. (16) and Harms et al. (14) found a four- to fivefold increase in lung lymph flow and an increase in Clymph/Cp in IL-2-treated sheep. We cannot account for the difference between their data and ours except to suggest that unilateral cautery of diaphragmatic lymphatics in their surgical preparations may have been inadequate to prevent some contribution of abdominal (liver) lymph to CMN efferent flow. We always cauterize lymphatics on both halves of the diaphragm (26). In previous studies, Douguet et al. (4) and Lei et al. (18) found an increase in splanchnic lymph flow and in splanchnic Clymph/Cp in IL-2-treated sheep.

The sheep treated chronically with IL-2 for 7 days showed many of the same signs and systemic effects as patients treated with IL-2: malaise, anorexia, fever, oliguria, hypotension, and tachycardia. However, the sheep did not develop anasarca or gain weight as some patients do. Our non-volume-loaded sheep did not develop edema, showed no evidence of vascular leakiness (normal plasma escape rates), and lost 15% of body weight. Decreased oral intake, resulting in an empty rumen and intestine, and a hypermetabolic state manifested as fever, tachycardia, and increased ventilation (all of these may account for weight loss despite a positive measured fluid balance) are the principal causes of the weight loss. The sheep also showed a decrease rather than an increase in cardiac output compared with humans.

In contrast, the volume-loaded sheep lost only 5% of their body weight over 3 days. These sheep developed histological evidence of pulmonary and intestinal edema and had an increased cardiac output during volume loading. Important physiological differences between healthy sheep and patients with malignancies who are receiving multiple medications account for some of the differences between our data and patient data. We conclude that the fluid management of subjects undergoing IL-2 treatment is critical in determining the edemagenic effects of the therapy.

Fluid management during IL-2 therapy is critical because of the concurrent development of hypoalbuminemia and myocardial dysfunction. Low plasma protein osmotic pressure combined with increased hydrostatic pressure unbalances the Starling forces in favor of extravascular liquid transudation. Intravascular volume loading further exacerbates extravascular liquid accumulation. More recently, treatment of hypotension and oliguria in patients undergoing IL-2 therapy with vasopressor drugs rather than with volume expansion has resulted in fewer cardiopulmonary complications including pulmonary edema. The studies by Gaynor et al. (12) in patients and Zielender et al. (36) in sheep demonstrate the benefits of vasopressor over volume support.

After intensive investigation of the pulmonary microvascular barrier by several methods, we conclude that IL-2 causes minimal or no injury to the lungs of sheep. Pulmonary edema during IL-2 therapy is caused primarily by hemodynamic imbalances in the lung.

ACKNOWLEDGEMENTS

The authors thank Michael Grady, Oscar Osorio, Pam Holland, and Richard Shanks for technical assistance and Julien Hoffman and Dennis Fisher for assistance with statistical analysis.

FOOTNOTES

   This study was supported by National Heart, Lung, and Blood Institute Program Project Grant HL-25816 and Grant HL-40891. Cetus Corporation, Emeryville, CA, provided additional support and all of the interleukin-2.

   E. H. Jerome is a recipient of the American Society of Anesthesiologists Young Investigator Award. D. Douguet was supported by the Institut National de la Sante et de la Recherche Medicale, France.

   Present address and address for reprint requests: E. H. Jerome, Dept. of Anesthesia, The New York Hospital-Cornell Medical Center, 525 East 68th St., New York, NY 10021.

Received 23 May 1994; accepted in final form 21 May 1996.

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