Tyloxapol attenuates the pathologic effects of endotoxin in rabbits and mortality following cecal ligation and puncture in rats by blockade of endotoxin receptor-ligand interactions.

We have previously demonstrated that the detergent Tyloxapol is effective in preventing reactions to endotoxin. We studied the effects of Tyloxapol on the morbidity and mortality from endotoxemia in rabbits and on the mortality in rats with sepsis. The effects of Tyloxapol on endotoxin binding and macrophage activation were studied in the macrophage cell line RAW264.7 and CHO cells expressing CD14. Isolated human leukocytes were used to study the effects of Tyloxapol on immune reactions, leukocyte motility, and phagocytosis. Intravenous Tyloxapol (200 mg/kg), given prior to or at the time of endotoxin infusion protected rabbits from developing shock. In rats with peritoneal sepsis, a lipid-rich diet and Tyloxapol given at the time of induction of peritonitis protected them from septic death. In vitro, Tyloxapol blocked the binding of endotoxin to murine macrophages and CHO cells expressing CD14, activation of macrophages, and also some antigen-antibody immune reactions (mediated by CD2, CD4, CD22, HLA-DR). Tyloxapol may prevent the reaction to endotoxin by desensitizing endotoxin-recognizing receptors.

Detergent inhibits 70-90% of responses to intravenous endotoxin in awake sheep.

Sheep have reactive pulmonary intravascular macrophages, which are essential for the marked pulmonary vascular response to infusions of small quantities of endotoxin. In another species with reactive pulmonary intravascular macrophages, horses, our laboratory found that an intravenous biosafe detergent, tyloxapol, inhibited some systemic and pulmonary responses to endotoxin (Longworth KE, Smith BL, Staub NC, Steffey EP, and Serikov V. Am J Vet Res 57: 1063-1066, 1996). We determined whether the same detergent would inhibit endotoxin responses in awake sheep. In 10 awake, instrumented sheep with chronic lung lymph fistulas, we did a control experiment by intravenously infusing 1 microg/kg Escherichia coli endotoxin. One week later, we gave 40 micromol/kg tyloxapol intravenously 1-4 h before infusing the same dose of endotoxin. In these paired studies, we compared pulmonary hemodynamics, lung lymph dynamics, body temperature, circulating leukocyte concentrations, and circulating tumor necrosis factor for 6 h. In all 10 sheep, tyloxapol blocked 80-90% of the pulmonary responses and 70-90% of the systemic responses. Tyloxapol is safe, inexpensive, easy to use, and effective immediately. It may be a clinically useful approach to contravening many of the effects of endotoxemia, in humans as well as animals.

Intravascular macrophage depletion attenuates endotoxin lung injury in anesthetized sheep.

We recently showed that we can selectively and safely deplete most (average 85%) of the pulmonary intravascular macrophages in sheep by intravenously infusing liposomes containing dichloromethylene bisphosphonate. After a 1-h stable baseline, we made a 6-h comparison after a 30-min intravenous endotoxin infusion (1 microg/kg) between six anesthetized control lambs and six anesthetized lambs in which the intravascular macrophages had been depleted 24 h previously. Three of the control lambs had been macrophage depleted and allowed to recover their intravascular macrophage population for >/=2 wk. After depletion, both the early and late pulmonary arterial pressure rises were dramatically attenuated. Our main interest, however, was in the acute lung microvascular injury response. The early and late rises in lung lymph flow and the increase in lung lymph protein clearance (lymph flow x lymph-to-plasma protein concentration ratio) were >90% attenuated. We conclude the pulmonary intravascular macrophages are responsible for most of the endotoxin-induced pulmonary hypertension and increased lung microvascular leakiness in sheep, although the unavoidable injury of other intravascular macrophages by the depletion regime may also contribute something.

Airway thermal volume in humans and its relation to body size.

The objective of this study was to investigate the influence of volume ventilation (VE) and cardiac output (Q) on the temperature of the expired gas at the distal end of the endotracheal tube in anesthetized humans. In 63 mechanically ventilated adults, we used a step decrease in the humidity of inspired gas to cool the lungs. After change from humid to dry gas ventilation, the temperature of the expired gas decreased. We evaluated the relationship between the inverse monoexponential time constant of the temperature fall (1/tau) and either VE or Q. When VE was increased from 5.67 +/- 1.28 to 7.14 +/- 1.60 (SD) l/min (P = 0. 02), 1/tau did not change significantly [from 1.25 +/- 0.38 to 1.21 +/- 0.51 min-1, P = 0.81]. In the 11 patients in whom Q changed during the study period (from 5.07 +/- 1.81 to 7.38 +/- 2.45 l/min, P = 0.02), 1/tau increased correspondingly from 0.89 +/- 0.22 to 1. 52 +/- 0.44 min-1 (P = 0.003). We calculated the airway thermal volume (ATV) as the ratio of the measured values Q to 1/tau and related it to the body height (BH): ATV (liters) = 0.086 BH (cm) - 9. 55 (r = 0.90).

Effect of particles on sheep lung hemodynamics parallels depletion and recovery of intravascular macrophages.

We previously showed in newborn lambs that the pulmonary hemodynamic responses to foreign particulate matter (liposomes; Monastral blue) developed in parallel with the maturation of the pulmonary intravascular macrophage system. We now report our use of the liposome-encapsulated heavy-metal-chelating agent dichloromethylene diphosphonate to deplete the intravascular macrophages of small lambs. Functionally and by quantitative histology, we depleted the vast majority of the intravascular macrophages (71% by Monastral blue particle retention, n = 22; 77% by histology; n = 2). Depletion success increased to > 90% as we optimized the liposome-depletion regime. Recovery of the lung hemodynamic response began within 3 days. By 2 wk, the functional responses had fully recovered (n = 8), and, according to quantitative histology, the macrophage population (n = 2) had recovered 65%. Macrophage depletion in lambs is relatively inexpensive and easy to achieve. It is a safe procedure and is followed by full recovery in approximately 2 wk, provided that an aseptic technique is used to prevent bacteremia.

Ultrastructural quantification of pulmonary intravascular macrophages in newborn and 2-week-old lambs.

BACKGROUND: Pulmonary intravascular macrophages are resident cells in the pulmonary circulation of sheep. Sheep, unlike species without pulmonary intravascular macrophages, exhibit pulmonary hypertension in response to intravenously injected particles. We reported in lambs that pulmonary vascular reactivity to intravenous particles increases with age as the population of intravascular macrophages develops. Preliminary quantitative histologic data showed that newborn lambs are born with few intravascular macrophages, but a large population develops over 2 weeks after birth. In this study, we present a complete quantitative analysis at the ultrastructural level. METHODS: We fixed five newborn and five 2-week-old lamb lungs by vascular perfusion and examined the tissue by electron microscopy. RESULTS: The fraction of capillary lumen taken up by intravascular macrophages/ monocytes is about three times greater in the lungs of 2-week-old lambs than that in newborn lambs (16% vs. 5%; P < 0.05). The fraction of capillary surface density associated with intravascular macrophages/monocytes is about three times greater in 2-week-old lambs than that in newborn lambs (8% vs. 3%; P < 0.05). The number of macrophages more than doubles with age (16 +/- 4 vs. 7 +/- 2; P < 0.05) and the estimated size (volume-weighted mean volume) increases by more than 1.5 times (294 +/- 46 microns 3 vs. 184 +/- 29 microns 3; P < 0.05). CONCLUSIONS: These data agree closely with Monastral blue retention by the lung (reported previously); there are more than twice as many mature pulmonary intravascular macrophages at 2 weeks than at 1 day after birth, and the cells are 1.5 times larger.

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

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 and 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.

Use of detergent to prevent initial responses to endotoxin in horses.

OBJECTIVE: To determine whether a detergent can prevent most of the early effects of i.v. infusion with Escherichia coli endotoxin (< 100 ng/kg of body weight) in horses: marked pulmonary hypertension, acute leukopenia, and fever. ANIMALS: 8 healthy adult horses (4 male, 4 female), 415 to 615 kg. DESIGN AND PROCEDURE: Control and detergent experiments were performed in each horse while it was awake but sedated. In control experiments, 10 to 100 ng of E coli endotoxin/kg was given. In detergent experiments, 100 mg of detergent/kg was given 1 hour before injecting endotoxin, similar to the control experiments. RESULTS: In control experiments, pulmonary arterial pressure increased transiently over 40 minutes by 33 +/- 8 mm of Hg (mean +/- SD; P < 0.001), then returned to baseline. Circulating leukocytes decreased to 47 +/- 19% (P < 0.02) of baseline by 1 hour after endotoxin, then increased above baseline by 6 hours. Rectal temperature increased by 0.7 +/- 0.4 C (P < 0.01). In detergent experiments, the increase in pulmonary arterial pressure was much less than that in the control experiments (8 +/- 7 mm of Hg; P < 0.001). Circulating leukocytes did not decrease, and the increase in rectal temperature after endotoxic was blocked. CONCLUSIONS: This attenuation of te response to endotoxin may occur because the normal steps in the response of pulmonary intravascular macrophages (ie, endocytosis of endotoxin and subsequent release of inflammatory mediators) are altered by the detergent. This low-technology, inexpensive, and safe treatment could be an important new clinical tool for veterinarians in combating endotoxemia.

Pulmonary intravascular macrophages in horses and ponies.

Seven horses (4 anesthetized and 3 awake) and 2 ponies (anesthetized) were studied to evaluate the high sensitivity of the pulmonary circulation of the horse to various blood-borne particles, and to establish the presence of intravascular macrophages in the lung. Pulmonary and systemic pressures and cardiac output before and during particle injection were measured in some animals. An anesthetized foal had a large increase in pulmonary arterial pressure (32 and 34 mm of Hg) within 1 minute of IV administration of small test doses of radioactively labeled liposomes (2.5 mumol/kg of body weight) or a 1% suspension of blue pigment (0.3 ml/kg), respectively. Quantitative real-time gamma camera imaging of the foal revealed high retention of the labeled liposomes during the first pass through the lungs; retention persisted throughout the experiment. Postmortem analysis revealed 55 and 47% lung retention of liposomes and blue pigment, respectively. The 2 anesthetized ponies had increased pulmonary artery pressure of 34 +/- 7 mm of Hg, decreased cardiac output, and 42% lung retention after administration of 1% blue pigment (0.2 ml/kg), whereas 3 awake horses had increased pressure of 28 +/- 9 mm of Hg after 1.8 x 10(8) (1.8-microns-diameter) latex microspheres/kg. None of the injected particles caused vascular obstruction, and they do not cause pulmonary vascular reactivity in species that lack pulmonary intravascular macrophages. Finally, 3 horses (1 anesthetized and 2 awake) were infused IV with small doses of the blue pigment, and their lungs were perfusion-fixed to identify specific labeling of the pulmonary intravascular macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of interleukin-2 on the pulmonary microvasculature in anesthetized sheep.

Interleukin-2 (IL-2) is reputed to cause pulmonary microvascular injury. We studied the pulmonary and splanchnic microcirculation of anesthetized sheep after one dose (1.8 x 10(6) IU/kg) of IL-2 (n = 9) and after six doses (1.8 x 10(6) IU.kg-1.dose-1) of IL-2 over 3 days (n = 9). Seven control sheep received only 5% dextrose diluent. We measured hemodynamics and lymph dynamics in anesthetized sheep after the final dose of IL-2 or diluent. After one dose of IL-2, caudal mediastinal node (mainly pulmonary) lymph flow was stable, whereas thoracic duct lymph flow increased from a baseline of 54 +/- 6 to 124 +/- 22 ml/h. After 3 days of IL-2, the caudal mediastinal node lymph flow increased from 7.7 +/- 5.5 to 19.0 +/- 14.8 ml/h 5-6 h after the final dose of IL-2, and thoracic duct lymph flow increased from 84 +/- 43 to 143 +/- 42 ml/h. The lymph-to-plasma protein concentration ratio increased after IL-2 for thoracic duct but not for caudal mediastinal node lymph. The equilibration rate of 125I-albumin from plasma to caudal mediastinal node lymph did not change, whereas plasma-to-thoracic duct lymph equilibration was faster after both one dose and 3 days of IL-2. Positron emission tomography showed no increase in the pulmonary transcapillary escape rate for 68Ga-labeled transferrin or in extravascular lung water (n = 4). We conclude that IL-2 at doses two to three times those used clinically does not significantly injure the pulmonary microcirculation of sheep.

Pulmonary hypertensive response to rabbit blood components in goats: role of thromboxane.

Transfusion of small quantities of heterologous blood may cause severe pulmonary hypertensive response in certain species. To determine the responsible component in the donor blood and the main mediator, we studied the responses of goats to small quantities of rabbit blood components and observed the effects of several pharmacologic agents on these responses. In anesthetized goats, a bolus injection of 0.004 ml/kg rabbit blood caused the pulmonary arterial pressure to increase from 25.3 +/- 2.8 to 57.1 +/- 11.6 cm H2O within 45 to 90 s, and the aortic thromboxane concentration rose from 44 +/- 38 to 238 +/- 104 pg/ml. Pulmonary vascular resistance increased more than 4-fold, whereas systemic vascular resistance increased moderately (50%). The erythrocyte stroma, mainly cell membranes, caused similar responses; other blood components were all ineffective. By blocking the production of thromboxane, indomethacin and U63557A (thromboxane synthetase inhibitor) abolished nearly all of the hemodynamic responses to rabbit blood. Isoproterenol also largely attenuated the responses to rabbit blood by blocking thromboxane production without interfering with the responses to the thromboxane mimic U46619. Nitrendipine (calcium-channel blocker) equally attenuated rabbit blood and U46619-induced hemodynamic responses but did not block thromboxane production. Chlorpheniramine (H1-receptor antagonist) partially blocked the hemodynamic responses to rabbit blood without affecting thromboxane production or U46619-induced responses. We conclude that the erythrocyte membrane is the responsible component in the donor blood and thromboxane is the predominant mediator. The main action of isoproterenol is to reduce thromboxane production and histamine participates by possible interaction with cyclooxygenase products.

Development of pulmonary intravascular macrophage function in newborn lambs.

We sought to determine whether pulmonary intravascular macrophages are involved in pulmonary vascular sensitivity to intravenously injected particles in sheep. We estimated that newborn lambs have few of these macrophages at 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 atrial pressure) after injection of either liposomes [2 +/- 3 (SD) cmH2O; n = 5] or Monastral blue particles (3 +/- 2 cmH2O; n = 6) and showed no net pulmonary production of thromboxane B2, the stable metabolite of the vasoconstrictor thromboxane A2. In contrast, five of those lambs 2 wk later showed both an increase in pulmonary vascular driving pressure after injection of liposomes and Monastral blue (20 +/- 16 and 25 +/- 15 cmH2O, respectively; P < 0.05) and net pulmonary production of thromboxane B2 (171 +/- 103 and 429 +/- 419 pg/ml plasma, respectively; P < 0.05). Older lambs (n = 5) had higher pulmonary uptakes than newborn lambs (n = 6) of radioactive liposomes (47 +/- 13 vs. 12 +/- 10%; P < 0.01) and Monastral blue (53 +/- 6 vs. 21 +/- 10%; P < 0.05). We conclude that pulmonary intravascular macrophages are responsible for the sensitivity of sheep to intravenous foreign particles and are essential for a cascade of processes leading to microvascular injury.

Downregulation of blood and bone marrow neutrophils decreases expression of acute lung injury in sheep.

We have shown that infusion of zymosan-activated plasma (ZAP) in sheep causes acute lung injury and downregulates peripheral blood neutrophils in that elicited superoxide release is reduced for at least 24 h after the infusion. The present study was designed to test the following hypotheses: 1) peripheral blood neutrophils are representative of neutrophils marginated in the pulmonary circulation, 2) blood neutrophils are downregulated because neutrophils developing in bone marrow are similarly affected, and 3) downregulated neutrophils have a reduced capacity to produce tissue injury. In a series of experiments in 21 sheep, we showed that elicited superoxide release was similar in peripheral blood neutrophils and in marginated neutrophils washed out of the pulmonary vascular bed. Measurements of superoxide release from blood and bone marrow neutrophils collected 2-24 h after ZAP infusion revealed progressive downregulation with time and greater downregulation of superoxide release in bone marrow neutrophils compared with peripheral blood neutrophils. Finally, after downregulating peripheral blood neutrophils, subsequent infusion of ZAP in conscious sheep produced sequestration of neutrophils in the pulmonary circulation but failed to produce a sustained increase in lung lymph protein clearance. The results suggest that neutrophil downregulation, as measured in vitro, is expressed in vivo as reduced ability of neutrophils to produce tissue injury when challenged by an activating agent.

Reliability of extravascular lung thermal volume measurements by thermal conductivity technique in sheep.

We tested the accuracy, sensitivity, and reproducibility of a new lung water computer, based on the thermal conductivity technique, in 22 anesthetized closed-chest ventilated sheep with different treatments: 1) controls (n = 8), 2) 0.05 ml/kg of oleic acid + 100 ml/kg of lactated Ringer solution (n = 6), and 3) airway instillation of saline [3.1 +/- 1.3 (SD) g/kg, n = 8]. After 4 h, we determined the extravascular lung water gravimetrically. We found a significant overall correlation between the final extravascular lung thermal volume and the gravimetric extravascular lung mass (P < 0.001). Although the average ratio of extravascular lung thermal volume to extravascular lung mass was 0.97 +/- 0.25 ml/g for all groups, the computer overestimated extravascular lung mass in controls by 10% (17 g) and underestimated it in sheep with oleic acid by 15% (95 g) and in sheep with airway instillation by 8% (37 g). The computer also underestimated the small quantities of saline placed via the airway in the alveolar space by 75% (61 g). Reproducibility of three consecutive measurements was 4.3% (SE). We conclude that the thermal conductivity technique has an ability to detect the baseline extravascular lung mass but has a poor ability to detect an accurate increment of the extravascular lung water under poor tissue perfusion in anesthetized ventilated sheep.

Effect of interstitial edema on lung lymph flow in goats in the absence of filtration.

We tested the effect of interstitial edema on lung lymph flow when no filtration occurred. In 16 anesthetized open-thorax ventilated supine goats, we set pulmonary arterial and left atrial pressures to nearly zero and measured lymph flow for 3 h from six lungs without edema and ten with edema. Lymph flow decreased exponentially in all experiments as soon as filtration ceased. In the normal lungs the mean half time of the lymph flow decrease was 12.7 +/- 4.8 (SD) min, which was significantly shorter (P less than 0.05) than the 29.1 +/- 14.8 min half time in the edematous lungs. When ventilation was stopped, lymph flow in the edematous lungs decreased as rapidly as in the normal lungs. The total quantity of lymph after filtration ceased was 2.7 +/- 0.8 ml in normal lungs and 9.5 +/- 6.3 ml in edematous lungs, even though extravascular lung water was doubled in the latter (8.4 +/- 2.4 vs. 3.3 +/- 0.4 g/g dry lung, P less than 0.01). Thus the maximum possible clearance of the interstitial edema liquid by the lymphatics was 6.3 +/- 4.8%. When we restarted pulmonary blood flow after 1-2 h in four additional goats, lymph flow recovered within 30 min to the baseline level. These findings support the hypothesis that lung lymph flow originates mainly from alveolar wall perimicrovascular interstitial liquid and that the contribution of the lung lymphatic system to the clearance of interstitial edema (bronchovascular cuffs, interlobular septa) is small.

Application of respiratory heat exchange for the measurement of lung water.

A noninvasive method for measuring pulmonary blood flow and lung mass (called airway thermal volume), based on the measurements of lung heat exchange with environment, is described. The lungs function as a steady-state heat exchange system, having an inner heat source (pulmonary blood flow) and an external heat sink (ventilation). Sudden changes in the steady-state condition, such as caused by hyperventilation of dry air, lead to a new steady state after a few minutes. The expired air temperature difference between the initial and final steady states is proportional to pulmonary blood flow, whereas the rate at which the new steady state is achieved is proportional to airway thermal volume. The method was tested in 20 isolated dogs lungs, 9 perfused goat lungs, and 27 anesthetized sheep. The expired air temperature fall during hyperventilation was inversely proportional to the perfusion rate of the isolated lungs, and half-time of the temperature fall was proportional to the lung tissue mass. Experiments in anesthetized sheep showed that the measured airway thermal volume is close to the total mass of the excised lungs, including its residual blood (r = 0.98). Pulmonary edema and fluid instillation into the bronchial tree increased in the measured lung mass.

Pulmonary hemodynamic reaction to foreign blood in goats and rabbits.

We have found that the goat is extraordinarily sensitive to very small quantities of rabbit or rat blood. As little as 0.004 ml/kg induces transient pulmonary hypertension [maximal rise in pulmonary arterial pressure 32 +/- 10 (SD) cmH2O] in goats. We hypothesized that this reaction may be related to the presence of the resident population of intravascular macrophages that reside in the pulmonary capillaries of goats. If that is so, then rabbits or rats, which have few or no intravascular macrophages, should not be reactive to foreign blood. We compared pulmonary hemodynamics and changes in blood thromboxane B2 concentrations among goats, rabbits, and rats in response to graded doses of foreign blood. The pulmonary reaction to foreign blood was much greater in goats than in rabbits or rats, even though we injected up to 10- or 60-fold larger amounts into the latter species. In goats the pulmonary vascular pressure response to rabbit blood was dose dependent in goats and correlated well with changes in systemic arterial thromboxane B2 concentrations [change in pulmonary arterial pressure = 0.07 (thromboxane B2) + 8.3, r = 0.79]. We also tested the prostaglandin H2 endoperoxide analogue (U-46619) and found that the goats are somewhat more reactive than rabbits. We conclude that the pulmonary hemodynamic reaction to foreign blood is consistent with the concept that the foreign erythrocytes are reacting with the pulmonary intravascular macrophages in goats. The lower reactivity of the rabbit pulmonary circulation to thromboxane may also have a role.