- Email: [email protected]
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
0272-5231/96 $0.00
THE LUNG IN LIVER DISEASE
+ .20
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE Shih-Wen Chang, MD, and Narumi Ohara, MD
Pulmonary intravascular phagocytosis, the clearance of circulating blood particulates by lung cells, is observed routinely in certain animal species, including sheep, horses, and goats. In these animals, the lung uptake of colloids, endotoxin, and bacteria from circulation is attributable to the large number of intravascular macrophages in the pulmonary microcirculation. The cell biology, interspecies variations, and pathophysiologic significance of these pulmonary intravascular macrophages (PIMs) are described elsewhere in this issue. In species lacking PIM, including humans, pulmonary intravascular phagocytosis is a rare finding. In these animals, the reticuloendothelial system is concentrated in the liver and spleen. Pulmonary intravascular phagocytosis has been reported sporadically in the setting of acute or chronic liver diseases in humans, but its clinical significance is unknown. This article reviews the clinical and experimental evidence supporting the hypothesis that chronic liver disease induces the development of PIM-like cells in species that normally do not contain PIM, and that these PIM-like cells are responsible for the observed intravascular phagocytosis. We first review some of the experimental models available for the study of liver-lung interactions in animals and present some data from our own studies in rats with biliary cirrhosis. We then summarize the experimental studies
that indicate that acquired liver disease in the rat leads to the appearance of intravascular macrophages in the pulmonary circulation along with the development of physiologically significant pulmonary intravascular phagocytosis. The clinical reports suggesting a similar process in humans with liver diseases are reviewed. Finally, we discuss some of the possible mechanisms for PIM induction and the potential pathophysiologic consequences of pulmonary intravascular phagocytosis. ANIMAL MODELS OF HEPATOPULMONARY SYNDROME The two most widely studied animal models of liver cirrhosis are the carbon tetrachloride/phenobarbital model19,38 and the chronic bile duct ligation Studies of the portal and systemic hemodynamics have been published using both of these animal mode l ~ .19,~ 31, , 38 In 1992, we published the first and, so far, the only study of the pulmonary circulation in rats with liver cirrhosis.1oWe chose the chronic bile duct ligation model over the carbon tetrachloride model to avoid any potential direct toxic effects of the chemical on the pulmonary vasculature. Hemodynamic Studies in Rats with Biliary Cirrhosisio Male, Sprague-Dawley rats were anesthetized with pentobarbital and randomized to
From the Midwest Consultants of Internal Medicine, Janesville, Wisconsin _____
_____
~
CLINICS IN CHEST MEDICINE
-
VOLUME 17 NUMBER 1 * MARCH 1996
137
138
CHANG & OHARA
common bile duct ligation and excision (BDL) or sham operation (control),consisting of laparotomy followed by closure of the incision. BDL rats became jaundiced within the first week following surgery and developed histologic evidence of biliary cirrhosis by the fourth week. BDL and time-matched control rats were studied at 4 to 5 weeks after surgery. In addition, rats were studied 10 days after surgery to control for the effect of cholestasis. Cholestatic rats exhibit hyperbilirubinemia to a degree similar to that of cirrhotic rats, but their livers show no gross or microscopic changes of cirrhosis. Systemic and pulmonary hemodynamic parameters were measured in awake, unanesthetized rats 24 to 48 hours following surgical implantation of vascular catheters in the aorta and main pulmonary artery.l* Cardiac outputs were measured by dye-dilution technique and vascular resistances were calculated accordingly. After obtaining baseline hemodynamic data, rats were exposed to hypoxic gas (8% oxygen) to elicit hypoxic pulmonary vasoconstriction. This was followed by infusion of angiotensin I1 to assess pulmonary vascular reactivity (Fig. 1). Table 1 shows the baseline hemodynamic data and hypoxic pressor response for the three study groups. There were no differences in baseline heart rate or mean aortic and pulmonary artery pressures in these rats. Cirrhotic rats exhibited increased cardiac index, however, so the calculated systemic as well Catheter Implantation
as pulmonary vascular resistances were decreased significantly. Of note, the hemodynamic data in cholestatic rats were similar to those in control rats, indicating that the observed effects were caused by cirrhosis and not cholestasis alone. Arterial hypoxemia is common in patients with liver disease and the cause may be multifactorial.3°,47 Severe hypoxemia associated with intrapulmonary shunting is the hallmark of the hepatopulmonary syndrome, as described in the article on that condition in this issue. In our study of rats with biliary obstruction, we documented increased alveolar-to-arterial (A-a) oxygen gradient in cirrhotic but not cholestatic rats (Fig. 2), suggesting an acquired abnormality in pulmonary gas exchange with the development of cirrhosis. In some of these cirrhotic rats, the A-a gradient remained elevated when the patient breathed 100% oxygen. This suggests that functional intrapulmonary shunting may contribute to the arterial hypoxemia in these animals. So far, we have not assessed anatomic intrapulmonary shunting with either microbubbles or macroaggregated albumin in this animal model. Hypoxic Pulmonary Vasoconstriction in Cirrhosis
Hypoxic pulmonary vasoconstriction (HPV) is a unique vascular response of the intrapul-
I
wore study ABG
1Wh 02
8% 0 2
I
P////////1
ABG
I
Plasma for liver chemistries Uver wight and histology
Figure 1. Hemodynamic study protocol in awake rats with biliary cirrhosis. Approximate time points (in minutes) are indicated. Following baseline hemodynamic measurement (heart rate, aortic pressure, pulmonary arterial pressure), study rats were exposed to hypoxic gas (8% OJ, infusion of angiotensin II (All), and hyperoxia (100% OJ. Cardiac output (asterisks) was performed by a dye dilution technique. ABG = arterial blood gas. (From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145798, 1992; with permission.)
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
139
Table 1. BASELINE HEMODYNAMIC DATA AND HYPOXIC PRESSOR RESPONSE Cirrhotic (n = 8)
Control ( n = 8)
HR (min-l) Pao (mm Hg) PPa (mm Hg) CI (mUminute/lOO g BW) TSR (mm Hg/minute/100 g BW/L) TPR (mm Hg/minute/lOO g BW/L) HPR-1 (mm Hg) HPR-2 (mm Hg) HPR-3 (mm Hg)
343 119.6 18.7 33.2 3694 577 13.1 12.3 11.0
+ 14 + 0.7 + 1.8 + 270 + 22 + 1.9 + 2.0 + 1.7 + 4.0
366 108.6 18.3 42.7 2628 455 4.8 3.6 4.7
+ 17 + 6.8 + 1.4 + 2.8' + 264' + 42* + 0.6' + 1.2* + 1.1*
Cholestatic (n = 6) 392 117.2 20.3 33.9 3502 670 14.1 11.9 11.8
+ 11 + 2.3 + +
+ + + + +
1.4 1.8 211 56 3.4 2.8 2.9
HR = Heart rate; Pao = mean aortic pressure: Ppa = mean pulmonary artery pressure: CI = cardiac index; BW = body weight: TSR = total systemic resistance: TPR = total pulmonary resistance; HPR = hypoxic pressor response. Values are mean + standard error of the mean. 'P<0.05 different from control value. From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis: An animal model of the hepatopulmonary syndrome. Am Rev Respir Dis 145:798, 1992;with permission.
monary microvessels to alveolar hypoxia.ss This mechanism is important in the optimal matching of perfusion to ventilation. Loss or depression of HPV has been reported in patients with liver c i r r h ~ s i s39, ' ~48~ and may con-
t
E
t *
g
IT 20/
8 1 4
8
-
8 -
i
10
0
A
Control
Cirrhotic
D
i
Cholestatic
-
i
~
0
Control
Cirrhotic
Cholestatic
Figure 2. A, Alveolar-arterial 0, gradient while breathing room air (RA). 6, Alveolar-arterial 0, gradient while breathing 100% 0,. *P<0.05 compared with control or cholestatic. (Adapted from Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145:798, 1992; with permission.)
tribute to the occurrence of hypoxemia. We found that rats with BDL-induced liver cirrhosis exhibit depression of HPV analogous to patients with severe liver disease.'O This is evident when HPV is expressed as either the change in mean pulmonary arterial pressures with hypoxic ventilation (see Table 1) or as the change in total pulmonary vascular resistance (Fig. 3). When lungs are removed from rats with biliary cirrhosis and perfused ex vivo with normal blood, the depression of HPV persists (Figs. 4 and 5), indicating that this abnormality is intrinsic to the pulmonary circulation of cirrhotic rats and is not caused by the depressive effect of circulating plasma factors. The fact that angiotensin 11-induced vasoconstriction was intact or enhanced (see Fig. 5 B ) suggests that this vasodepressor effect of cirrhosis is relatively stimulus-specific and not caused by generalized paralysis of vascular smooth muscle. Interestingly, angiotensin I1 infusion appears to enhance subsequent HPV in isolated perfused lungs from cirrhotic rats (see Fig. 5A). The mechanism of this angiotensin I1 effect is unknown but may relate to stimulation of cyclic-guanosine monophosphate or adenosine monophosphate hydrolysis.33,51 One of our findings, which has not been adequately investigated, is the apparent hypoxic vasodilation in some perfused lungs from cirrhotic rats. This is seen in rat lungs that exhibit vascular instability and mild edema accumulation during baseline perfusion. With onset of hypoxic ventilation, the pulmonary arterial pressure flattens out or
140
CHANG & OHARA Control
Cirrhotic
Cholestatic
1
300
-
T
E
&, 200 -
I
E E
100 I-
k0.05
0
RA
8% o2
RA
8% 0 2
RA
8% o2
Figure 3. Hypoxic pulmonary vasoconstriction in unanesthetized rats. Individual values for total pulmonary vascular resistance (TPR) before (RA = room air) and during hypoxic ventilation (8% 0,) are shown. (From Chang S,Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145798, 1992; with permission.)
decreases, followed by a rapid rise with return to normoxia. It is likely that this pradoxic response of the pulmonary circulation to hypoxia represents the extreme of vascular injury.
Vascular Permeability Changes in Liver Cirrhosis Several observations from our initial studies of this animal model suggested the possibility that pulmonary endothelial cell injury, with increased vascular permeability, may be a central defect in the pulmonary circulatory
I. /L
/
C
10 min
Time
response to liver cirrhosis. These observations include: (1)increased wet lung-to-body weight ratio in cirrhotic rats,'" (2) increased susceptibility of perfused lungs from cirrhotic rats to edema during ex vivo perfusion,'O and (3) the similarity in pulmonary vascular reactivity to animals with acute lung vascular injury attributable to endotoxin.12 Subsequently, we studied pulmonary vascular permeability in vivo using a double-label techniq~e.~ lZ5Iodine-albumin and 51chromate-erythrocytes were injected into rat tail veins and allowed to circulate for 1 hour. At the end of that period, the rats were euthanized and the
Figure 4. Representative pulmonary arterial tracings of isolated, perfused lungs from control (A) and biliary cirrhotic rats (B and C). The lungs are perfused at constant speed with blood from normal rats, and vasoconstriction was induced by ventilating the lung with hypoxic gas (3% 0,) or injection with angiotensin II (All). The lungs from rat B lost hypoxic pulmonary vasoconstriction but preserved angiotensin II vasoconstriction. The lungs from rat C exhibited unstable pulmonary arterial pressure associated with development of lung edema. A paradoxic vasodilation is observed with hypoxic ventilation (C). (From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145:798, 1992; with permission.)
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
T
m
I
E E c
.-m
Li
I"' 10 n P
a
n "
#1
A
#3 #4 Hypoxic Response
#2
#5
1
"
B
INTRAVASCULAR PHAGOCYTOSIS IN EXPERIMENTAL LIVER DISEASE
Bile Duct Ligation Model
20
40
141
0.05
0.1
Angiotensin II (pg)
Figure 5. Sequential hypoxic (A) and All (B) pressor responses in perfused lungs from control (open bar) and cirrhotic rats (hatched bar). APP = change in pulmonary artery perfusion pressure. (From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145798, 1992; with permission.)
lungs removed. We found that extravascular lung leak index, a measure of in vivo pulmonary vascular permeability, was elevated markedly in cirrhotic rats (Fig. 6 ) . In addition, the bloodless wet-to-dry weight ratio of the lung, a measure of lung water content, was elevated significantly in cirrhotic rats.9In support of the conclusion from the physiologic parameters of lung vascular permeability, pulmonary endothelial cell injury was demonstrated by electronmicroscopy in lungs from cirrhotic ratsy Interestingly, cholestatic rats (10 days after BDL) showed no evidence of such injury and had levels of lung albumin index and lung wet-to-dry ratio comparable to In a related we found that biliary cirrhosis leads to increased albumin leak index in lung and kidney, but not in heart or brain, of cirrhotic rats. It is tempting to link the increased vascular permeability in certain organs to the development of organ dysfunction in cirrhosis, but this association remains speculative.
In examining the electronmicrographs of lung sections from rats with biliary cirrhosis, we were struck by the large number of large mononuclear cells within small intrapulmonary vessels. These were not seen in shamoperated controls. These cells, in morphology, resemble the PIMs found in sheep and pigs. Because PIMs are not usually present in rat lungs, this suggests the possibility that biliary obstruction or liver cirrhosis induces PIM formation in the rat. To determine whether these intravascular cells in cirrhotic rats possess the phagocytic capacity of PIMs, we compared the lung uptake of fluorescent latex particles (1 pm in diameter) and technetium-99 (y9mTc)-labeled microaggregated albumin in cirrhotic and sham-operated rats.* Two hours after intravascular injection of fluorescent latex particles, lungs from control rats contained very few individual latex particles on fluorescent microscopy, whereas lungs from cirrhotic rats showed clusters of latex particles within large intravascular mononuclear cells (Fig. 7). On electronmicroscopy, these cells exhibit morphologic features of mature macrophages, in-
21
:
+ 0 0 0
i l P
l
I
Control
I
Cirrhotic
Figure 6. Lung albumin leak index in control and biliary cirrhotic rats. Lung vascular permeability is assessed by injecting 1251-alb~min and %r-tagged erythrocytes into tail vein and counting blood and lung radioactivity 1 hour later. Lung albumin leak index = (extravascular lung lZ5lcounts/blood 1 2 5 1 - ~ per ~ ~g)/lung n t ~ blood wt. Lung blood counts in lung/Wr counts in blood/blood weight = sample wt.
142
CHANG & OHARA
Figure 7. Light microscopic sections of lungs from bile duct ligation (BDL) and control rats. A, Control rat lung: small vessels containing very few leukocytes. 6,Lung from BDL rats (4 weeks): large number of leukocytes are observed in the vessel lumen. Two large, macrophage-like cells with injected latex particles are present (arrow). There is also perivascular edema (sfar). Bar marker = 10 pm. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 94:2009, 1994; with permission of The American Society for Clinical Investigation.)
cluding indented nucleus, abundant cytoplasm containing phagocytic vacuoles, and many irregular surface projections (Fig. 8). Although fluorescent latex particles allow localization and identification of the cell(s) responsible for intravascular phagocytosis, it is difficult to quantitate the number of particles taken up by these cells for the purpose of comparison between animal and animal and experimental and control groups. We
therefore used a method commonly used in clinical nuclear medicine studies with a radioactive marker (99mTc) labeled to small, intravascular particles that are taken up from circulation by the reticuloendothelial system. Instead of performing external imaging with a gamma camera, we removed the organ of interest and measured its radioactivity ex vivo. This allows an accurate calculation of the phagocytic capacity of that organ. For the
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
143
Figure 8. Electronmicroscopic section of lung from BDL rats. M = intravascular macrophage; N = neutrophil; E = erythrocyte. The intravascular macrophage contains ingested latex particles (arrow). Bar marker = 1 pm. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 942009, 1994; with permission of The American Society for Clinical Investigation.)
phagocytic stimulus, we used commercially available microaggregated albumin (Microlite), which has a mean particle diameter of 1 km. Because of its ease of preparation and reproducible particle size, this agent is superior to the sulfur colloids used in liver-spleen scans in humans.49 One hour after intravascular injection of 99mTc-Microlite,the lungs were removed for measurement of radioactivity. Lung 9 9 m T ~ Microlite uptake is low in sham-operated control rats (<1%)and increased with time following bile duct ligation (Fig. 9). The lung uptake in cirrhotic rats averaged about %YO, representing a 47-fold increase compared with the corresponding value in control rats. Our data showed that the extent of pulmonary intravascular phagocytosis is correlated with the severity of liver disease, as reflected by the increase in spleen weight (Fig. 10).
passively were filtered into lung interstitium during periods of acute lung vascular injury by CCL. Although this was not addressed specifically, there apparently was no signifi-
Time After Surgery (W)
Carbon Tetrachloride Model In studying the phagocytic activity of Kupffer cells after acute liver injury with carbon tetrachloride (CCl,), Paumgartner et a P reported increased intravascular uptake of carbon in the lung during the acute phase of liver cell necrosis, 1 to 2 days after CCl, administration. It is unclear whether these carbon particles were taken up by lung cells or
Figure 9. Lung 9 9 m T ~ - M i ~ ruptake ~ l i t e after BDL. Data shown are mean (and SE bars) lung uptake as a percentalbuage of total injected dose of 99mTc-microaggregated min (Microlite). Rats were studied at 1, 2, and 4 weeks after BDL or sham-surgery (CONT). ' k 0 . 0 5 compared with control values at the correspondingtime point. Open bar = sham-surgery; hatched bar = bile duct ligation. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 94:2009, 1994; with permission of The American Society for Clinical Investigation.)
144
CHANG & OHARA
10''
0
1
2
3
Spleen Weight (9)
Figure 10. Correlation of lung 99mTc-Microlite uptake with spleen weight in BDL rats. Lung 9 9 m T ~ - M i ~ r uptake, ~lite a measure of pulmonary intravascular phagocytosis, is significantly correlated with spleen weight, a reflection of the severity and chronicity of cholestatic liver disease. Regression line: Log(Lung 99mTc-MicroliteUptake) = 0.731 (spleen wt)-0.347, r = 0.87; P < 0.01.W = 1 week; o = 2 weeks; V = 4 weeks. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 94:2009, 1994; with permission of The American Society for Clinical Investigation.)
cant uptake of carbon by the lung at later periods, when CC1,-induced liver cirrhosis was well established. More recently, Noda et al'" studied Kupffer cell function using 99mTc-millimicrosphered albumin in CC1,-induced chronic liver injury. They reported that, with CC14-induced liver cirrhosis, the liver uptake of 99mTc-millimicrosphered albumin decreased from 80% to 42%, whereas lung uptake increased eight-fold, from 2.3% to 18%. Although no morphologic study was done to clarify which cell(s) was responsible for the increased lung particle uptake in CCh-induced liver cirrhosis, this study does suggest that the induction of pulmonary intravascular phagocytosis is not limited to the BDL model.
The Role of Pulmonary lntravascular Macrophages in Pulmonary lntravascular Phagocytosis in Chronic Liver Disease We have used the term PIM loosely to describe the large mononuclear cells found in the pulmonary circulation of our cirrhotic rats because the cells in question resemble the
resident cell population found in sheep and pig lungs in morphology and function. We recognize (and expect) that there are some differences between the cell population induced in rats via a pathologic process and the normal resident cell population of healthy sheep. The presence of adhesion complex with the underlying endothelial cell, for example, has been considered an important feature of PIM in sheep.57To date, we have not identified adhesion complexes between the PIM-like cells and endothelial cells, although many areas of tight apposition are seen. In the only report of PIMs in human lung, adhesion complex with endothelial cells also was lacking.16 Another important point to emphasize is the presence of other inflammatory cells in the pulmonary microcirculation of cirrhotic rats. These include neutrophils, lymphocytes, monocytes, and cells with morphology intermediate between monocytes and intravascular macro phage^.'^ In preliminary studies quantitating the uptake of latex particles by various cells in the pulmonary circulation, however, it appears that intravascular macrophages, by far, are the predominant cells in lung particle uptake.13
Role of Pulmonary lntravascular Macrophages in CirrhosisAssociated Pulmonary Circulatory Alterations Whether the induction of the PIM-like cells contributes to the pulmonary circulatory changes observed in liver cirrhosis remains uncertain. Figure 11 shows a hypothetical schema that incorporates PIMs and their eicosanoid metabolite, thromboxane AZ, in the pathogenesis of the pulmonary circulatory abnormalities in liver cirrhosis. Although simplistic, the schema is consistent with all our experimental observations in the BDL model of liver cirrhosis."'O We postulated that liver cirrhosis induces the formation of PIMs in the lung microcirculation by an as yet unknown mechanism (discussed subsequently). Thromboxane AZ, a cyclooxygenase metabolite of arachidonic acid, is released by activated macrophages, including PIMs.~Thromboxane A, is a potent vasoconstrictor with potential direct effects on pulmonary vascular permeability.62Other vasoactive metabolites of PIMs, including prostaglandins, leukotrienes, and platelet ac-
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
Liver Cirrhosis
Pulmonary lntravascular Macrophage
I
Thromboxane A2
Increased Lu na Vascular Permeabilitv
t I
Lung Edema
Depression of HPV Impaired Gas Exchange Figure 11. A hypothetical schema for pulmonary circulatory alterations in cirrhosis. HPV = hypoxic pulmonary vasoconstriction. (From Chang S, Ohara N: Increased pulmonary vascular permeability in rats with biliary cirrhosis: Role of thromboxane A2. Am J Physiol 264:L245, 1993; with permission.)
tivating factor: all may participate in synergistic interactions with thromboxane A, to cause pulmonary endothelial cell injury and l1 increased lung vascular ~ermeability.~, Pulmonary interstitial edema resulting from an increase in pulmonary vascular permeability was documented in rats with biliary cirrhosis by both physiologic measurements (lung wet-to-dry weight ratio and albumin leak index) and ultrastructural observations? A previous study by Furukawa et alZoalso reported finding pulmonary interstitial edema in rats with CC1,-induced liver cirrhosis. Regardless of the specific mechanisms involved in lung microvascular injury, increased vascular permeability and interstitial edema often lead to depression of hypoxic pulmonary vasoconstriction40and impaired gas exchange. The resultant hypoxemia may be ascribed to the abnormal ventilationperfusion matching as well as intrapulmonary shunting. POTENTIAL MECHANISMS FOR INDUCTION OF PULMONARY INTRAVASCULAR MACROPHAGES IN LIVER CIRRHOSIS
145
study from our laboratory also suggests that the PIM-like cells in cirrhotic rats likely are originating from blood monocytes14 and are not simply migrating Kupffer cells.8Vascular perfusion of lungs from BDL rats yields a large number of monocytes early in the course of BDL. As liver disease progresses, cells with morphology intermediate between monocytes and macrophages are recovered. Even in rats with well-established liver cirrhosis, the population of PIM-like cells remains a small minority. The majority of the intravascular cells in the pulmonary circulation are composed of activated mon~cytes.'~ Of note, recent reports are in agreement with our observation. First, monocytes isolated from peripheral blood of cirrhotic subjects exhibit in vitro evidence of activation.I7 Second, mice with biliary obstruction have elevated levels of cytokines, including tumor necrosis factor and interleukin. These results indicate the presence of activated macrophages in these cirrhotic animals2 Role of Lipopolysaccharide Bacterial endotoxin, or lipopolysaccharide (LPS), is a potent activator of circulating leukocytes and a central mediator of sepsis-induced lung i n j ~ r yAdministration .~ of endotoxin induces monocyte retention in rabbit and the appearance of PIM-like cells in rat lungs.58In addition, pulmonary intravascular phagocytosis has been noted in rats following endotoxemia.26,46 Because endogenous endotoxemia may occur in cirrhosis,22,45 it is reasonable to speculate that chronic endotoxemia may contribute to the appearance of PIMs in liver cirrhosis. In additional experiments in the rat, we were able to document acute effects of LPS on pulmonary intravascular phagocytosis as measured by lung 99mTc-Microlite~ p t a k e . ~ This effect of LPS was transitory, however, and repeated injection of LPS for 4 weeks failed to induce PIM formation (Chang S, Ohara N, unpublished observation, 1993). We believe that chronic endotoxemia alone cannot account for the induction of PIM in the setting of biliary cirrhosis.
Cell of Origin
Role of Estrogen
It generally is accepted that PIMs are derived from circulating monocytes.61A recent
Liver cirrhosis is known to cause an imbalance in the estrogen-anti-estrogen status of
146
CHANG & OHARA
lungsz1 and the absence of lung uptake of the body, in favor of the estrogens.' Mikhael 99n'Tc-sulfur colloids during liver-spleen and Evens36reported that estrogen treatment scans. In analogy to studies in experimental of guinea pigs and toads leads to mobilization animals, however, it is possible that PIMs of large numbers of phagocytic cells from may be inducible in humans under patholiver, spleen, and bone marrow into the intralogic conditions. In addition to one report vascular space. These cells then are trapped showing PIM-like cells in resected lung speciin the pulmonary microcirculation but retain men,16a series of case reports in nuclear medtheir ability to phagocytize colloidal particles. icine literature has suggested that pulmonary These authors suggest that increased levels of intravascular phagocytosis can occur in huestrogenic hormones stimulate the migration mans.23, 24, 27. 28 of reticuloendothelial cells to the lung and Dehring and Wismarlh studied electronmitherefore account for the occasional observasections of lung specimen resected tion of increased lung uptake of 9 9 m T ~ - ~ ~ l fcroscopic ur colloids in patients with liver disease.30 for various noninfectious indications. In 7 of 40 specimens, they were able to identify intraApplying our modern concept of the monovascular cells with morphology resembling nuclear phagocyte system to these results, PIMs in sheep, including the presence of their findings are consistent with estrogenphagocytic vacuoles and micropinocytosis induced stimulation of marrow-derived vermiformis. Junctional complexes between monocytes, with subsequent pulmonary sethese intravascular macrophages and endoquestration of the activated m o n ~ c y t e sIt . ~is~ thelial cells were not noted, however. The not clear whether prolonged stimulation with intravascular macrophages in these human estrogenic hormones would cause the activilung specimens were not quantitated, but ated monocytes to develop into intravascular they seemed less plentiful than the PIMs in macrophages. In addition to its potential effects on the Keyes et a124reported a series of 22 patients mononuclear phagocyte system, estrogen with marked increases in lung uptake of treatment has been shown to reduce hypoxic 9 9 m T ~ - ~ ~colloid l f u r during routine liver impulmonary vasoconstriction in the sheep aging. These cases represent 1.6% of all the lung.60Although this effect is small in magniliver scans performed in a 33-month period. tude, alterations in estrogen hormone levels In each case, technical factors that might lead in cirrhosis could contribute to the changes to increased lung uptake from microemboliin pulmonary vascular reactivity.1° zations were excluded. The most common diagnosis (found in ll patients) in this group Other Potential Mechanisms of patients was some form of malignancy. Of the 22 patients, 18 had some evidence of liver Because PIMs are derived from circulating abnormality, but only four had documented monocytes, cytokine factors that regulate the liver cirrhosis. It is noteworthy that the findgrowth and differentiation of monocytes and ing of marked lung uptake of ""Tc-sulfur monocyte precursor cells are likely to be imcolloid was associated with high mortality, portant in the induction of PIM in liver cirwith a median survival of only 3.5 months rhosis. One such factor is the macrophage after the liver scan.24 colony stimulating factor (CSF-l), which inIn a subsequent study, Klingensmith28reduces macrophage proliferation and differenported on 27 patients with increased lung t i a t i ~ n CSF-1 . ~ ~ content has been reported to uptake of 99mTc-sulfur colloid during routine be elevated in human liver disease.18Whether liver-spleen scans. A variety of disease proincreased activity of CSF-1 plays a role in the cesses existed in this group, including 12 cancellular alterations in the lung in rats with cers, six collagen vascular diseases, four biliary cirrhosis is unknown. Laennec's cirrhosis, and three infections. The survival in this group of patients was significantly better than in the group reported by PULMONARY INTRAVASCULAR Keyes et al.24Various other reports in the PHAGOCYTOSIS IN HUMANS nuclear medicine literaturez3,27, 52, 53 further support the association between increased It generally is accepted that normal human lung uptake of ""Tc-sulfur colloid and liver lungs do not contain PIMs. This belief is disease and malignancy, as well as other less based on lack of finding intravascular macrocommon disorder^.^ phages on morphometric studies of human
PULMONARY INTRAVASCULAR PHAGOCYTOSISIN LIVER DISEASE Table 2. CONDITIONS ASSOCIATED WITH PULMONARY INTRAVASCULAR PHAGOCYTOSIS Common Malignancy (breast carcinoma, lymphoma) Liver disease (cirrhosis, metastatic tumor, abscess) Infection (abscess) Uncommon Histiocytosis X Mucopolysaccharidosis type II Organ transplantation (bone marrow, liver) from Chang S: Pulmonary intravascular inflammation in sepsis and liver disease. Semin Respir Crit Care Med 15463, 1994; with permission.
The mechanism for the observed pulmonary intravascular phagocytosis in humans is unknown. Based on animal studies discussed earlier, we might speculate that the presence of intravascular macrophages or activated monocytes in the pulmonary microcirculation is responsible for the intense lung uptake of intravascular colloid particles. If so, this would indicate that PIMs could be induced in humans by some pathophysiologic stimuli associated with liver disease or malignancy. Much work remains to be done to clearly demonstrate the presence of PIMs in these settings and to elucidate the cellular and molecular mechanisms of the PIM induction. CLINICAL CONSEQUENCES OF PULMONARY INTRAVASCULAR PHAGOCYTOSIS Clearance of lntravascular Particles
Warner and Brain57 discussed potential functions of PIM in species (such as sheep and pigs) that have these cells in large numbers. In such animals, PIMs are an important part of the mononuclear phagocyte system and participate in the reticuloendothelial clearance of blood-borne particles. Using quantitative magnetometry, Brain et a157demonstrated that greater than 80% of injected iron oxide particles are removed from circulation by PIMs in the lungs of sheep, calves, and cats. PIMs also may help in removal of effete erythrocytes, given that erythrophagocytosis by PIMs has been reported by several 61
In addition to clearance of intravascular particles, PIMs also remove circulating bacteria from the blood stream, thereby participating in host defense against septicemia. Lungs containing PIMs also show increased uptake of circulating LPS, although cellular localiza-
147
tion of LPS within PIMs has not been demonstrated conclusively. Not unexpectedly, uptake of endotoxin by sheep lung is followed rapidly by inflammatory cell infiltration and signs of acute lung vascular injury.59 Pulmonary Hypertension
Although the incidence is relatively low, severe pulmonary hypertension is associated with a variety of chronic liver diseases in humans (discussed elsewhere in this issue). It is tempting to speculate that PIMs may play a role in the pathogenesis of this disorder because animals with PIMs are uniquely susceptible to severe pulmonary hypertension in response to intravascular particles and endotoxin. Miyamoto et a137showed that injections of liposomes in sheep cause dose-dependent rises in pulmonary arterial pressure mediated by thromboxane. The same response occurs with injection of monastral blue, a colloidal pigment.33In vitro, isolated PIMs from pig lungs metabolize arachidonic acid into various metabolites, including thromboxane B2.3 We also have documented increased thromboxane B, level in lung tissue from rats with biliary cirrhosis, and suggested that thromboxane A, may contribute to the increase in lung vascular permeability in these animals? In ex vivo perfused lungs from rats with biliary cirrhosis, we have found increased pulmonary hypertensive response to endotoxin and phorbol myristate acetate.56 The hypothesis that PIMs may play a role in the development of pulmonary hypertension will be very difficult to prove or disprove. The incidence of pulmonary hypertension in cirrhosis is low and there has not been any report of pulmonary intravascular phagocytosis occurring in the setting of pulmonary hypertension. Moreover, we have not noted any animals with pulmonary hypertension in our hemodynamic study of rats with biliary cirrhosis.1o Adult Respiratory Distress Syndrome in Liver Cirrhosis
Adult respiratory distress syndrome (ARDS) is characterized by acute lung vascular injury, increased vascular permeability, and noncardiogenic pulmonary edema. In 1987, Matuschak et a135reported that patients
148
CHANG & OHARA
with end-stage liver failure are highly susceptible to the development of sepsis-induced ARDS. Twenty-three (79%) of the 29 patients with end-stage liver failure requiring intensive care unit admission developed ARDS, whereas only 3 (6.8%) of 44 patients without liver failure developed ARDS. Once initiated, the ARDS was irreversible, with mortality of 100% in this series. ARDS also complicates the clinical course of patients undergoing liver transplantation. Takaoka et a154found 10 cases of ARDS in 198 patients undergoing orthotopic liver transplantation. The mortality in these patients with liver disease approached 8070, much higher than the 40% to 60% mortality quoted for ARDS patients in general. In a recent case report,% ARDS, occurring in the setting of liver allograft rejection, resolved promptly following hepatic retransplantation, suggesting that the presence of liver disease somehow contributes to the development and perpetuation of ARDS. Various theories have been proposed to explain the increased susceptibility of patients with chronic liver disease to ARDS (discussed elsewhere in this issue). Matuschak%,35 described four elements of the systemic inflammatory response that are under hepatic regulation: (1) clearance of endotoxin, bacteria, and vasoactive substances by hepatic Kupffer cells, (2) hepatic production of cytokine and eicosanoid mediators, (3) metabolic inactivation and detoxification of these mediators by Kupffer cell-hepatocyte interaction, and (4) hepatic synthesis of acute-phase protein, which moderates cytokine-mediated inflammation and tissue injury. It has been suggested that liver failure leads to the derangement of one or more of these mechanisms, thereby predisposing these patients to ARDS. Pulmonary lntravascular Phagocytosis and Adult Respiratory Distress Syndrome
The results of our recent study in rats with biliary cirrhosis suggest another potential mechanism to explain ARDS in patients with liver cirrhosis-the induction of PIMs. The comparative studies in different animal species have shown that sensitivity to endotoxin-induced lung injury correlates with the presence of intravascular macrophages. Animals with PIMs (sheep, pigs) are exquisitely sensitive to endotoxin, showing response to
microgram quantities, with severe pulmonary hypertension, increased vascular permeability, and lung edema. In contrast, animals without PIMs (rat, mice) can tolerate up to milligram quantities of endotoxin and, when they die of septic shock, their lungs usually are free of significant edema. In rats with biliary cirrhosis, we found both a quantitative and qualitative change in the pulmonary circulatory response to bacterial endotoxin" As the lungs acquired large numbers of intravascular macrophages, the rats became extremely endotoxin-sensitive.Shamoperated control rats tolerated up to 1 mg/ kg of endotoxin without any obvious adverse effects, but rats with biliary cirrhosis showed increased lethargy and piloerection with as little as 0.01 mg/kg of endotoxin. The median lethal dose for endotoxin was shifted by at least two log orders of magnitude in rats with biliary cirrhosis.8 Moreover, whereas control rats dying after high doses of endotoxin had normal lung weights, rats with biliary cirrhosis dying after low doses of endotoxin had markedly increased wet lung weights and lung wet-to-dry weight ratios, indicating the development of severe pulmonary edema? In another recent study, Kleber et alZ5 showed that endotoxin infusion causes marked reductions in cardiac index and renal blood flow in rats with biliary cirrhosis but not in sham-operated rats. Unfortunately, pulmonary circulatory effects of endotoxin were not measured in that study. Based on the results of these animal studies, as well as clinical reports of pulmonary intravascular phagocytosis in certain patients with chronic liver disease, we propose the following hypotheses: (1) liver cirrhosis induces the development of intravascular macrophages in the pulmonary circulation and (2) the presence of large numbers of PIMs predisposes these patients to develop ARDS following sepsis or endotoxemia. Certainly, additional clinical and experimental studies are necessary to confirm or refute these hypotheses. ARDS is a vexing clinical problem associated with high morbidity and mortality. Although certain predisposing medical conditions have been well described, ARDS occurs unpredictably among patients with common risk factors. It is clear that host factors are likely to be important in determining individual susceptibility to ARDS, yet none of these factors has been identified clearly. PIMs, by virtue of their unique location, can be stimu-
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
lated by circulating bacteria, endotoxin, or cytokines to release additional vasoactive mediators, such as tumor necrosis factor, eicosanoids, and reactive oxygen intermediates, directly into the pulmonary microcirculation. The resultant amplification of the systemic inflammatory response in the pulmonary microcirculation may lead to lung microvascular injury and the clinical syndrome of ARDS.
References 1. Adlercreutz H. Hepatic metabolism of estrogen in health and disease. N Engl J Med 290:1981, 1974 2. Bemelmans MHA, Gouma DJ, Greve JW, et a1 Cytokines tumor necrosis factor and interleukin-6 in experimental biliary obstruction in mice. Hepatology 15:1132, 1992 3. Bertram TA, Overby LH, Danilowicz R, et al: Pulmonary intravascular macrophages metabolize arachidonic acid in vitro. Am Rev Respir Dis 138:936, 1988 4. Bomzon A, Rosenberg M, Gali D, et al: Systemic hypotension and decreased pressor response in dogs with chronic bile duct ligation. Hepatology 6:595, 1986 5. Brigham KL, Meyrick B: Endotoxin and lung injury. Am Rev Respir Dis 133:913, 1986 6. Caramelo C, Femandez-Gallardo S, Santos JC, et al: Increased levels of platelet-activating factor in blood from patients with cirrhosis of the liver. Eur J Clin Invest 177,1987 7. Chane S: Pulmonarv intravascular inflammation in sepsizand liver disease. Semin Respir Crit Care Med 15463, 1994 8. Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 942009, 1994 9. Chang S, Ohara N Increased pulmonary vascular permeability in rats with biliary cirrhosis: Role of thromboxane A,. Am J Physiol264:L245, 1993 10. Chang S, Ohara N Pulmonary circulatory dysfunction in rats with biliary cirrhosis: An animal model of the hepatopulmonary syndrome. Am Rev Respir Dis 145:798, 1992 11. Chang S, Westcott JY, Pickett WC, et al: Endotoxininduced lung injury in rats: role of eicosanoids. J Appl Physiol 66:2407, 1989 12. Chang S, Feddersen CO, Henson PM, et al: Plateletactivating factor mediates hemodynamic changes and lung injury in endotoxin-treated rats. J Clin Invest 79:1498, 1987 13. Chida M, Spom PHS, Wamer AE, et a 1 Phagocytic activity of pulmonary intravascular macrophages from rats with biliary cirrhosis [abstract]. Am J Respir Crit Care Med 149:A1028, 1994 14. Chida M, Spom PHS, Wang X, et al: Perfusion isolation of pulmonary intravascular macrophages from rats with biliary cirrhosis [abstract]. Am Rev Respir Dis 147A307, 1993 15. Daoud FS, Reeves JT, Schaefer J W Failure of hypoxic pulmonary vasoconstriction in patients with liver cirrhosis. J Clin Invest 51:1076, 1972 16. Dehring DJ, Wismar BL Intravascular macrophages in pulmonary capillaries of humans. Am Rev Respir Dis 139:1027, 1989
149
17. Deviere J, Content J, Denys C, et al: Excessive in vitro bacterial lipopolysaccharide-induced production of monokines in cirrhosis. Hepatology 11:628, 1990 18. Esposti SD, Stanley R, Zem MA: Macrophage-colony stimulating factor (CSF-1) content is markedly increased in human liver disease [abstract]. Hepatology 12:908, 1990 19. Femandez-Munoz D, Caramelo C, Santos JC, et a1 Systemic and splanchnic hemodynamic disturbances in conscious rats with experimental liver cirrhosis without ascites. Am J Physiol 249:G316, 1985 20. Furukawa T, Yasumoto K, Inokuchi K Pulmonary interstitial edema in experimental cirrhosis of the liver in rats. Eur Surg Res 16:366, 1984 21. Gehr PM, Bachofen M, Weibel E R The normal human lung. Ultrastructure and morphometric estimation of diffusion capacity. Respir Physiol32121,1978 22. Gouma DJ, Coelho JCU, Fisher JD, et a1 Endotoxemia after relief of biliary obstruction by internal and external drainage in rats. Am J Surg 151:476, 1986 23. Imarisio JJ: Liver scan showing intense lung uptake in neoplasia and infection. J Nucl Med 16:188, 1975 24. Keyes JW Jr, Wilson GA, Quinones J D An evaluation of lung uptake of colloid during liver imaging. J Nucl Med 14:687, 1973 25. Kleber G, Braillon A, Gaudin C, et a1 Hemodynamic effects of endotoxin and platelet activating factor in cirrhotic rats. Gastroenterblogy 103:282, 1992 26. Klingensmith WC 111, Lovett VJ Jr: Lung uptake of "mTc-sulfur colloid secondary to intraperitoneal endotoxin. J Nucl Med 15:1028, 1974 27. Klingensmith WC 111, Ryerson TW, Corman JL: Lung uptake of 99mTc-sulfurcolloid in organ transplantation. J Nucl Med 14757,1973 28. Klingensmith WC 111, Yang SL, Wagner HN Jr: 99mTcsulfur colloid in liver and spleen imaging. J Nucl Med 19:31, 1978 29. Kountouras J, Billing BH, Scheuer PJ: Prolonged bile duct obstruction: A new experimental model for cirrhosis in the rat. Br J Exp Pathol 65305, 1984 30. Krowka MJ: Pulmonary aspects of chronic liver disease and liver transplantation. Mayo Clin Proc 60:407, 1985 31. Lee SS, Girod C, Braillon A, et al: Hemodynamic characterization of chronic bile duct-ligated rats: Effect of pentobarbital sodium. Am J Physiol 2 5 ~ ~ 1 7i986 6, I1 stimulates earlv 32. Liu F-Y. Coean MG: Aneiotensin " proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate. J Clin Invest 84:83, 1989 33. Longworth KE, Westgate AM, Grady MK, et al: Development of pulmonary intravascular macrophage function in newborn lambs. J Appl Physiol 73:2608, 1992 34. Matuschak GM, Shaw BW: Adult respiratory distress syndrome associated with acute liver allograft rejection: Resolution following hepatic retransplantation. Crit Care Med 15:878, 1987 35. Matuschak GM, Rinaldo JE, Pinsky MR, et al: Effect of end-stage liver failure on the incidence and resolution of the adult respiratory distress syndrome. J Crit Care 2162, 1987 36. Mikhael MA, Evens RG: Migration and embolization of macrophages to the lung: A possible mechanism for colloidal uptake in the lung during liver scanning. J Nucl Med 1622, 1975 37. Miyamoto K, Schultz E, Heath T, et al: Pulmonary intravascular macrophages and hemodynamic effects of liposomes in sheep. J Appl Physiol 641143, 1988 I
"
150
CHANG&OHARA
38. Murray BM, Paller M S Decreased pressor reactivity to angiotensin I1 in cirrhotic rats. Evidence of a postreceptor defect in angiotensin action. Circ Res 57424,1985 39. Naeije R, Hallemans R, Mols P, et a 1 Hypoxic pulmonary vasoconstriction in liver cirrhosis. Chest 80570, 1981 40. Newman JH, Loyd JE, English K, et al: Effect of 100% oxygen on lung vascular function in awake sheep. J Appl Physiol M1379, 1983 41. Noda T, Mimura H, Orita K Assessment of Kupffer cell function in rats with chronic liver injury caused by CCb. Hepatogastroenterology 37319, 1990 42. Ohara N, Voelkel NF, Chang S Tissue eicosanoids and vascular permeability in rats with chronic biliary obstruction. Hepatology 18:111, 1993 43. Ohgami M, Doerschuk CM, Gie RP, et a1 Late effect of endotoxin on the accumulation and function of monocytes in rabbit lungs. Am Rev Respir Dis 146:190, 1992 44. Paumgartner G, Longueville J, Leevy CM: Phagocytic activity in experimental liver injury. Exp Mol Pathol 9:161, 1968 45. Prytz H, Jolst-Christensen J, Korner B, et al: Portal venous and systemic endotoxemia in patients with cirrhosis. Scand J Gastroenterol 112357, 1976 46. Quinones JD. Localization by technetium-sulfur colloid after RES stimulation. J Nucl Med 1 4 4 3 , 1973 47. Rodman T, Sobel M, Close HP: Arterial oxygen unsaturation and the ventilation-perfusion defect of Laennec’s cirrhosis. N Engl J Med 263:73, 1960 48. Rodriquez-Roisin R, Roca J, Agusti AG, et al: Gas exchange and pulmonary vascular reactivity in patients with liver cirrhosis. Am Rev Respir Dis 135:1085, 1987 49. Saha GB, Feiglin DHI, O’Donnell JK, et al: Experience with 99mTechnetiumalbumin colloid kit for reticuloendothelial imaging. J Nucl Med Techno1 14149,1986 50. Sherr CJ: Regulation of mononuclear phagocyte pro-
51. 52. 53. 54. 55. 56.
57. 58.
59. 60. 61. 62.
liferation by colony-stimulating factor-1. Int J Cell Cloning 8(suppl 1):46, 1990 Smith JB, Lincoln TM: Angiotensin decreases cyclic GMP accumulation produced by atrial natriuretic factor. Am J Physiol 253C147, 1987 Stadalnik RC: Diffuse lung uptake of Tc-99m-sulfur colloid. Semin Nucl Med 10:106, 1980 Steinbach H L Pulmonary accumulation of 9ymTc-sulfur colloid during liver scanning. Texas Med 68:137, 1972 Takaoka F, Borwn MR, Paulsen AW, et al: Adult respiratory distress syndrome following orthotopic liver transplantation. Clin Transplantation 3:294,1989 Voelkel NF: Mechanisms of hypoxic pulmonary vasoconstriction. Am Rev Respir Dis 133:1186,1986 Wang X, Ohara N, Chang S Enhanced pulmonary hypertensive responses to endotoxin and phorbol myristate acetate in rats with chronic biliary obstruction [abstract]. Clin Res 40697A, 1992 Warner AE, Brain JD: The cell biology and pathogenic role of pulmonary intravascular macrophages. Am J Physiol 258L1, 1990 Warner AE, =amp MM Jr, Bellows CF, et al: Endotoxemia enhances lung uptake of circulating particles in species lacking pulmonary intravascular macrophages [abstract]. Am Rev Respir Dis 139:A158, 1989 Warner AE, Decamp MM Jr, Molina RM, et a1 Pulmonary removal of circulating endotoxin results in acute lung injury in sheep. Lab Invest 59:219, 1988 Wetzel RC, Zacur HA, Sylvester JT: Effect of puberty and estradiol on hypoxic vasomotor response in isolated sheep lungs. J Appl Physiol 56:1199, 1984 Winkler GC, Cheville NF: Monocytic origin and postnatal mitosis of intravascular macrophages in the porcine lung. J Leukocyte Biol 38:471, 1985 Yoshimura K, Todd ML, Pier KG, et a1 Effects of thromboxane A, analogue and prostacyclin on lung fluid balance in newborn lambs. Circ Res 65:1409, 1989
Address reprint requests to Shih-Wen Chang, MD Midwest Consultants of Internal Medicine 2640 Milton Avenue PO Box 505 Janesville, WI 53547-0505
THE LUNG IN LIVER DISEASE
+ .20
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE Shih-Wen Chang, MD, and Narumi Ohara, MD
Pulmonary intravascular phagocytosis, the clearance of circulating blood particulates by lung cells, is observed routinely in certain animal species, including sheep, horses, and goats. In these animals, the lung uptake of colloids, endotoxin, and bacteria from circulation is attributable to the large number of intravascular macrophages in the pulmonary microcirculation. The cell biology, interspecies variations, and pathophysiologic significance of these pulmonary intravascular macrophages (PIMs) are described elsewhere in this issue. In species lacking PIM, including humans, pulmonary intravascular phagocytosis is a rare finding. In these animals, the reticuloendothelial system is concentrated in the liver and spleen. Pulmonary intravascular phagocytosis has been reported sporadically in the setting of acute or chronic liver diseases in humans, but its clinical significance is unknown. This article reviews the clinical and experimental evidence supporting the hypothesis that chronic liver disease induces the development of PIM-like cells in species that normally do not contain PIM, and that these PIM-like cells are responsible for the observed intravascular phagocytosis. We first review some of the experimental models available for the study of liver-lung interactions in animals and present some data from our own studies in rats with biliary cirrhosis. We then summarize the experimental studies
that indicate that acquired liver disease in the rat leads to the appearance of intravascular macrophages in the pulmonary circulation along with the development of physiologically significant pulmonary intravascular phagocytosis. The clinical reports suggesting a similar process in humans with liver diseases are reviewed. Finally, we discuss some of the possible mechanisms for PIM induction and the potential pathophysiologic consequences of pulmonary intravascular phagocytosis. ANIMAL MODELS OF HEPATOPULMONARY SYNDROME The two most widely studied animal models of liver cirrhosis are the carbon tetrachloride/phenobarbital model19,38 and the chronic bile duct ligation Studies of the portal and systemic hemodynamics have been published using both of these animal mode l ~ .19,~ 31, , 38 In 1992, we published the first and, so far, the only study of the pulmonary circulation in rats with liver cirrhosis.1oWe chose the chronic bile duct ligation model over the carbon tetrachloride model to avoid any potential direct toxic effects of the chemical on the pulmonary vasculature. Hemodynamic Studies in Rats with Biliary Cirrhosisio Male, Sprague-Dawley rats were anesthetized with pentobarbital and randomized to
From the Midwest Consultants of Internal Medicine, Janesville, Wisconsin _____
_____
~
CLINICS IN CHEST MEDICINE
-
VOLUME 17 NUMBER 1 * MARCH 1996
137
138
CHANG & OHARA
common bile duct ligation and excision (BDL) or sham operation (control),consisting of laparotomy followed by closure of the incision. BDL rats became jaundiced within the first week following surgery and developed histologic evidence of biliary cirrhosis by the fourth week. BDL and time-matched control rats were studied at 4 to 5 weeks after surgery. In addition, rats were studied 10 days after surgery to control for the effect of cholestasis. Cholestatic rats exhibit hyperbilirubinemia to a degree similar to that of cirrhotic rats, but their livers show no gross or microscopic changes of cirrhosis. Systemic and pulmonary hemodynamic parameters were measured in awake, unanesthetized rats 24 to 48 hours following surgical implantation of vascular catheters in the aorta and main pulmonary artery.l* Cardiac outputs were measured by dye-dilution technique and vascular resistances were calculated accordingly. After obtaining baseline hemodynamic data, rats were exposed to hypoxic gas (8% oxygen) to elicit hypoxic pulmonary vasoconstriction. This was followed by infusion of angiotensin I1 to assess pulmonary vascular reactivity (Fig. 1). Table 1 shows the baseline hemodynamic data and hypoxic pressor response for the three study groups. There were no differences in baseline heart rate or mean aortic and pulmonary artery pressures in these rats. Cirrhotic rats exhibited increased cardiac index, however, so the calculated systemic as well Catheter Implantation
as pulmonary vascular resistances were decreased significantly. Of note, the hemodynamic data in cholestatic rats were similar to those in control rats, indicating that the observed effects were caused by cirrhosis and not cholestasis alone. Arterial hypoxemia is common in patients with liver disease and the cause may be multifactorial.3°,47 Severe hypoxemia associated with intrapulmonary shunting is the hallmark of the hepatopulmonary syndrome, as described in the article on that condition in this issue. In our study of rats with biliary obstruction, we documented increased alveolar-to-arterial (A-a) oxygen gradient in cirrhotic but not cholestatic rats (Fig. 2), suggesting an acquired abnormality in pulmonary gas exchange with the development of cirrhosis. In some of these cirrhotic rats, the A-a gradient remained elevated when the patient breathed 100% oxygen. This suggests that functional intrapulmonary shunting may contribute to the arterial hypoxemia in these animals. So far, we have not assessed anatomic intrapulmonary shunting with either microbubbles or macroaggregated albumin in this animal model. Hypoxic Pulmonary Vasoconstriction in Cirrhosis
Hypoxic pulmonary vasoconstriction (HPV) is a unique vascular response of the intrapul-
I
wore study ABG
1Wh 02
8% 0 2
I
P////////1
ABG
I
Plasma for liver chemistries Uver wight and histology
Figure 1. Hemodynamic study protocol in awake rats with biliary cirrhosis. Approximate time points (in minutes) are indicated. Following baseline hemodynamic measurement (heart rate, aortic pressure, pulmonary arterial pressure), study rats were exposed to hypoxic gas (8% OJ, infusion of angiotensin II (All), and hyperoxia (100% OJ. Cardiac output (asterisks) was performed by a dye dilution technique. ABG = arterial blood gas. (From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145798, 1992; with permission.)
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
139
Table 1. BASELINE HEMODYNAMIC DATA AND HYPOXIC PRESSOR RESPONSE Cirrhotic (n = 8)
Control ( n = 8)
HR (min-l) Pao (mm Hg) PPa (mm Hg) CI (mUminute/lOO g BW) TSR (mm Hg/minute/100 g BW/L) TPR (mm Hg/minute/lOO g BW/L) HPR-1 (mm Hg) HPR-2 (mm Hg) HPR-3 (mm Hg)
343 119.6 18.7 33.2 3694 577 13.1 12.3 11.0
+ 14 + 0.7 + 1.8 + 270 + 22 + 1.9 + 2.0 + 1.7 + 4.0
366 108.6 18.3 42.7 2628 455 4.8 3.6 4.7
+ 17 + 6.8 + 1.4 + 2.8' + 264' + 42* + 0.6' + 1.2* + 1.1*
Cholestatic (n = 6) 392 117.2 20.3 33.9 3502 670 14.1 11.9 11.8
+ 11 + 2.3 + +
+ + + + +
1.4 1.8 211 56 3.4 2.8 2.9
HR = Heart rate; Pao = mean aortic pressure: Ppa = mean pulmonary artery pressure: CI = cardiac index; BW = body weight: TSR = total systemic resistance: TPR = total pulmonary resistance; HPR = hypoxic pressor response. Values are mean + standard error of the mean. 'P<0.05 different from control value. From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis: An animal model of the hepatopulmonary syndrome. Am Rev Respir Dis 145:798, 1992;with permission.
monary microvessels to alveolar hypoxia.ss This mechanism is important in the optimal matching of perfusion to ventilation. Loss or depression of HPV has been reported in patients with liver c i r r h ~ s i s39, ' ~48~ and may con-
t
E
t *
g
IT 20/
8 1 4
8
-
8 -
i
10
0
A
Control
Cirrhotic
D
i
Cholestatic
-
i
~
0
Control
Cirrhotic
Cholestatic
Figure 2. A, Alveolar-arterial 0, gradient while breathing room air (RA). 6, Alveolar-arterial 0, gradient while breathing 100% 0,. *P<0.05 compared with control or cholestatic. (Adapted from Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145:798, 1992; with permission.)
tribute to the occurrence of hypoxemia. We found that rats with BDL-induced liver cirrhosis exhibit depression of HPV analogous to patients with severe liver disease.'O This is evident when HPV is expressed as either the change in mean pulmonary arterial pressures with hypoxic ventilation (see Table 1) or as the change in total pulmonary vascular resistance (Fig. 3). When lungs are removed from rats with biliary cirrhosis and perfused ex vivo with normal blood, the depression of HPV persists (Figs. 4 and 5), indicating that this abnormality is intrinsic to the pulmonary circulation of cirrhotic rats and is not caused by the depressive effect of circulating plasma factors. The fact that angiotensin 11-induced vasoconstriction was intact or enhanced (see Fig. 5 B ) suggests that this vasodepressor effect of cirrhosis is relatively stimulus-specific and not caused by generalized paralysis of vascular smooth muscle. Interestingly, angiotensin I1 infusion appears to enhance subsequent HPV in isolated perfused lungs from cirrhotic rats (see Fig. 5A). The mechanism of this angiotensin I1 effect is unknown but may relate to stimulation of cyclic-guanosine monophosphate or adenosine monophosphate hydrolysis.33,51 One of our findings, which has not been adequately investigated, is the apparent hypoxic vasodilation in some perfused lungs from cirrhotic rats. This is seen in rat lungs that exhibit vascular instability and mild edema accumulation during baseline perfusion. With onset of hypoxic ventilation, the pulmonary arterial pressure flattens out or
140
CHANG & OHARA Control
Cirrhotic
Cholestatic
1
300
-
T
E
&, 200 -
I
E E
100 I-
k0.05
0
RA
8% o2
RA
8% 0 2
RA
8% o2
Figure 3. Hypoxic pulmonary vasoconstriction in unanesthetized rats. Individual values for total pulmonary vascular resistance (TPR) before (RA = room air) and during hypoxic ventilation (8% 0,) are shown. (From Chang S,Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145798, 1992; with permission.)
decreases, followed by a rapid rise with return to normoxia. It is likely that this pradoxic response of the pulmonary circulation to hypoxia represents the extreme of vascular injury.
Vascular Permeability Changes in Liver Cirrhosis Several observations from our initial studies of this animal model suggested the possibility that pulmonary endothelial cell injury, with increased vascular permeability, may be a central defect in the pulmonary circulatory
I. /L
/
C
10 min
Time
response to liver cirrhosis. These observations include: (1)increased wet lung-to-body weight ratio in cirrhotic rats,'" (2) increased susceptibility of perfused lungs from cirrhotic rats to edema during ex vivo perfusion,'O and (3) the similarity in pulmonary vascular reactivity to animals with acute lung vascular injury attributable to endotoxin.12 Subsequently, we studied pulmonary vascular permeability in vivo using a double-label techniq~e.~ lZ5Iodine-albumin and 51chromate-erythrocytes were injected into rat tail veins and allowed to circulate for 1 hour. At the end of that period, the rats were euthanized and the
Figure 4. Representative pulmonary arterial tracings of isolated, perfused lungs from control (A) and biliary cirrhotic rats (B and C). The lungs are perfused at constant speed with blood from normal rats, and vasoconstriction was induced by ventilating the lung with hypoxic gas (3% 0,) or injection with angiotensin II (All). The lungs from rat B lost hypoxic pulmonary vasoconstriction but preserved angiotensin II vasoconstriction. The lungs from rat C exhibited unstable pulmonary arterial pressure associated with development of lung edema. A paradoxic vasodilation is observed with hypoxic ventilation (C). (From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145:798, 1992; with permission.)
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
T
m
I
E E c
.-m
Li
I"' 10 n P
a
n "
#1
A
#3 #4 Hypoxic Response
#2
#5
1
"
B
INTRAVASCULAR PHAGOCYTOSIS IN EXPERIMENTAL LIVER DISEASE
Bile Duct Ligation Model
20
40
141
0.05
0.1
Angiotensin II (pg)
Figure 5. Sequential hypoxic (A) and All (B) pressor responses in perfused lungs from control (open bar) and cirrhotic rats (hatched bar). APP = change in pulmonary artery perfusion pressure. (From Chang S, Ohara N: Pulmonary circulatory dysfunction in rats with biliary cirrhosis. Am Rev Respir Dis 145798, 1992; with permission.)
lungs removed. We found that extravascular lung leak index, a measure of in vivo pulmonary vascular permeability, was elevated markedly in cirrhotic rats (Fig. 6 ) . In addition, the bloodless wet-to-dry weight ratio of the lung, a measure of lung water content, was elevated significantly in cirrhotic rats.9In support of the conclusion from the physiologic parameters of lung vascular permeability, pulmonary endothelial cell injury was demonstrated by electronmicroscopy in lungs from cirrhotic ratsy Interestingly, cholestatic rats (10 days after BDL) showed no evidence of such injury and had levels of lung albumin index and lung wet-to-dry ratio comparable to In a related we found that biliary cirrhosis leads to increased albumin leak index in lung and kidney, but not in heart or brain, of cirrhotic rats. It is tempting to link the increased vascular permeability in certain organs to the development of organ dysfunction in cirrhosis, but this association remains speculative.
In examining the electronmicrographs of lung sections from rats with biliary cirrhosis, we were struck by the large number of large mononuclear cells within small intrapulmonary vessels. These were not seen in shamoperated controls. These cells, in morphology, resemble the PIMs found in sheep and pigs. Because PIMs are not usually present in rat lungs, this suggests the possibility that biliary obstruction or liver cirrhosis induces PIM formation in the rat. To determine whether these intravascular cells in cirrhotic rats possess the phagocytic capacity of PIMs, we compared the lung uptake of fluorescent latex particles (1 pm in diameter) and technetium-99 (y9mTc)-labeled microaggregated albumin in cirrhotic and sham-operated rats.* Two hours after intravascular injection of fluorescent latex particles, lungs from control rats contained very few individual latex particles on fluorescent microscopy, whereas lungs from cirrhotic rats showed clusters of latex particles within large intravascular mononuclear cells (Fig. 7). On electronmicroscopy, these cells exhibit morphologic features of mature macrophages, in-
21
:
+ 0 0 0
i l P
l
I
Control
I
Cirrhotic
Figure 6. Lung albumin leak index in control and biliary cirrhotic rats. Lung vascular permeability is assessed by injecting 1251-alb~min and %r-tagged erythrocytes into tail vein and counting blood and lung radioactivity 1 hour later. Lung albumin leak index = (extravascular lung lZ5lcounts/blood 1 2 5 1 - ~ per ~ ~g)/lung n t ~ blood wt. Lung blood counts in lung/Wr counts in blood/blood weight = sample wt.
142
CHANG & OHARA
Figure 7. Light microscopic sections of lungs from bile duct ligation (BDL) and control rats. A, Control rat lung: small vessels containing very few leukocytes. 6,Lung from BDL rats (4 weeks): large number of leukocytes are observed in the vessel lumen. Two large, macrophage-like cells with injected latex particles are present (arrow). There is also perivascular edema (sfar). Bar marker = 10 pm. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 94:2009, 1994; with permission of The American Society for Clinical Investigation.)
cluding indented nucleus, abundant cytoplasm containing phagocytic vacuoles, and many irregular surface projections (Fig. 8). Although fluorescent latex particles allow localization and identification of the cell(s) responsible for intravascular phagocytosis, it is difficult to quantitate the number of particles taken up by these cells for the purpose of comparison between animal and animal and experimental and control groups. We
therefore used a method commonly used in clinical nuclear medicine studies with a radioactive marker (99mTc) labeled to small, intravascular particles that are taken up from circulation by the reticuloendothelial system. Instead of performing external imaging with a gamma camera, we removed the organ of interest and measured its radioactivity ex vivo. This allows an accurate calculation of the phagocytic capacity of that organ. For the
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
143
Figure 8. Electronmicroscopic section of lung from BDL rats. M = intravascular macrophage; N = neutrophil; E = erythrocyte. The intravascular macrophage contains ingested latex particles (arrow). Bar marker = 1 pm. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 942009, 1994; with permission of The American Society for Clinical Investigation.)
phagocytic stimulus, we used commercially available microaggregated albumin (Microlite), which has a mean particle diameter of 1 km. Because of its ease of preparation and reproducible particle size, this agent is superior to the sulfur colloids used in liver-spleen scans in humans.49 One hour after intravascular injection of 99mTc-Microlite,the lungs were removed for measurement of radioactivity. Lung 9 9 m T ~ Microlite uptake is low in sham-operated control rats (<1%)and increased with time following bile duct ligation (Fig. 9). The lung uptake in cirrhotic rats averaged about %YO, representing a 47-fold increase compared with the corresponding value in control rats. Our data showed that the extent of pulmonary intravascular phagocytosis is correlated with the severity of liver disease, as reflected by the increase in spleen weight (Fig. 10).
passively were filtered into lung interstitium during periods of acute lung vascular injury by CCL. Although this was not addressed specifically, there apparently was no signifi-
Time After Surgery (W)
Carbon Tetrachloride Model In studying the phagocytic activity of Kupffer cells after acute liver injury with carbon tetrachloride (CCl,), Paumgartner et a P reported increased intravascular uptake of carbon in the lung during the acute phase of liver cell necrosis, 1 to 2 days after CCl, administration. It is unclear whether these carbon particles were taken up by lung cells or
Figure 9. Lung 9 9 m T ~ - M i ~ ruptake ~ l i t e after BDL. Data shown are mean (and SE bars) lung uptake as a percentalbuage of total injected dose of 99mTc-microaggregated min (Microlite). Rats were studied at 1, 2, and 4 weeks after BDL or sham-surgery (CONT). ' k 0 . 0 5 compared with control values at the correspondingtime point. Open bar = sham-surgery; hatched bar = bile duct ligation. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 94:2009, 1994; with permission of The American Society for Clinical Investigation.)
144
CHANG & OHARA
10''
0
1
2
3
Spleen Weight (9)
Figure 10. Correlation of lung 99mTc-Microlite uptake with spleen weight in BDL rats. Lung 9 9 m T ~ - M i ~ r uptake, ~lite a measure of pulmonary intravascular phagocytosis, is significantly correlated with spleen weight, a reflection of the severity and chronicity of cholestatic liver disease. Regression line: Log(Lung 99mTc-MicroliteUptake) = 0.731 (spleen wt)-0.347, r = 0.87; P < 0.01.W = 1 week; o = 2 weeks; V = 4 weeks. (From Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 94:2009, 1994; with permission of The American Society for Clinical Investigation.)
cant uptake of carbon by the lung at later periods, when CC1,-induced liver cirrhosis was well established. More recently, Noda et al'" studied Kupffer cell function using 99mTc-millimicrosphered albumin in CC1,-induced chronic liver injury. They reported that, with CC14-induced liver cirrhosis, the liver uptake of 99mTc-millimicrosphered albumin decreased from 80% to 42%, whereas lung uptake increased eight-fold, from 2.3% to 18%. Although no morphologic study was done to clarify which cell(s) was responsible for the increased lung particle uptake in CCh-induced liver cirrhosis, this study does suggest that the induction of pulmonary intravascular phagocytosis is not limited to the BDL model.
The Role of Pulmonary lntravascular Macrophages in Pulmonary lntravascular Phagocytosis in Chronic Liver Disease We have used the term PIM loosely to describe the large mononuclear cells found in the pulmonary circulation of our cirrhotic rats because the cells in question resemble the
resident cell population found in sheep and pig lungs in morphology and function. We recognize (and expect) that there are some differences between the cell population induced in rats via a pathologic process and the normal resident cell population of healthy sheep. The presence of adhesion complex with the underlying endothelial cell, for example, has been considered an important feature of PIM in sheep.57To date, we have not identified adhesion complexes between the PIM-like cells and endothelial cells, although many areas of tight apposition are seen. In the only report of PIMs in human lung, adhesion complex with endothelial cells also was lacking.16 Another important point to emphasize is the presence of other inflammatory cells in the pulmonary microcirculation of cirrhotic rats. These include neutrophils, lymphocytes, monocytes, and cells with morphology intermediate between monocytes and intravascular macro phage^.'^ In preliminary studies quantitating the uptake of latex particles by various cells in the pulmonary circulation, however, it appears that intravascular macrophages, by far, are the predominant cells in lung particle uptake.13
Role of Pulmonary lntravascular Macrophages in CirrhosisAssociated Pulmonary Circulatory Alterations Whether the induction of the PIM-like cells contributes to the pulmonary circulatory changes observed in liver cirrhosis remains uncertain. Figure 11 shows a hypothetical schema that incorporates PIMs and their eicosanoid metabolite, thromboxane AZ, in the pathogenesis of the pulmonary circulatory abnormalities in liver cirrhosis. Although simplistic, the schema is consistent with all our experimental observations in the BDL model of liver cirrhosis."'O We postulated that liver cirrhosis induces the formation of PIMs in the lung microcirculation by an as yet unknown mechanism (discussed subsequently). Thromboxane AZ, a cyclooxygenase metabolite of arachidonic acid, is released by activated macrophages, including PIMs.~Thromboxane A, is a potent vasoconstrictor with potential direct effects on pulmonary vascular permeability.62Other vasoactive metabolites of PIMs, including prostaglandins, leukotrienes, and platelet ac-
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
Liver Cirrhosis
Pulmonary lntravascular Macrophage
I
Thromboxane A2
Increased Lu na Vascular Permeabilitv
t I
Lung Edema
Depression of HPV Impaired Gas Exchange Figure 11. A hypothetical schema for pulmonary circulatory alterations in cirrhosis. HPV = hypoxic pulmonary vasoconstriction. (From Chang S, Ohara N: Increased pulmonary vascular permeability in rats with biliary cirrhosis: Role of thromboxane A2. Am J Physiol 264:L245, 1993; with permission.)
tivating factor: all may participate in synergistic interactions with thromboxane A, to cause pulmonary endothelial cell injury and l1 increased lung vascular ~ermeability.~, Pulmonary interstitial edema resulting from an increase in pulmonary vascular permeability was documented in rats with biliary cirrhosis by both physiologic measurements (lung wet-to-dry weight ratio and albumin leak index) and ultrastructural observations? A previous study by Furukawa et alZoalso reported finding pulmonary interstitial edema in rats with CC1,-induced liver cirrhosis. Regardless of the specific mechanisms involved in lung microvascular injury, increased vascular permeability and interstitial edema often lead to depression of hypoxic pulmonary vasoconstriction40and impaired gas exchange. The resultant hypoxemia may be ascribed to the abnormal ventilationperfusion matching as well as intrapulmonary shunting. POTENTIAL MECHANISMS FOR INDUCTION OF PULMONARY INTRAVASCULAR MACROPHAGES IN LIVER CIRRHOSIS
145
study from our laboratory also suggests that the PIM-like cells in cirrhotic rats likely are originating from blood monocytes14 and are not simply migrating Kupffer cells.8Vascular perfusion of lungs from BDL rats yields a large number of monocytes early in the course of BDL. As liver disease progresses, cells with morphology intermediate between monocytes and macrophages are recovered. Even in rats with well-established liver cirrhosis, the population of PIM-like cells remains a small minority. The majority of the intravascular cells in the pulmonary circulation are composed of activated mon~cytes.'~ Of note, recent reports are in agreement with our observation. First, monocytes isolated from peripheral blood of cirrhotic subjects exhibit in vitro evidence of activation.I7 Second, mice with biliary obstruction have elevated levels of cytokines, including tumor necrosis factor and interleukin. These results indicate the presence of activated macrophages in these cirrhotic animals2 Role of Lipopolysaccharide Bacterial endotoxin, or lipopolysaccharide (LPS), is a potent activator of circulating leukocytes and a central mediator of sepsis-induced lung i n j ~ r yAdministration .~ of endotoxin induces monocyte retention in rabbit and the appearance of PIM-like cells in rat lungs.58In addition, pulmonary intravascular phagocytosis has been noted in rats following endotoxemia.26,46 Because endogenous endotoxemia may occur in cirrhosis,22,45 it is reasonable to speculate that chronic endotoxemia may contribute to the appearance of PIMs in liver cirrhosis. In additional experiments in the rat, we were able to document acute effects of LPS on pulmonary intravascular phagocytosis as measured by lung 99mTc-Microlite~ p t a k e . ~ This effect of LPS was transitory, however, and repeated injection of LPS for 4 weeks failed to induce PIM formation (Chang S, Ohara N, unpublished observation, 1993). We believe that chronic endotoxemia alone cannot account for the induction of PIM in the setting of biliary cirrhosis.
Cell of Origin
Role of Estrogen
It generally is accepted that PIMs are derived from circulating monocytes.61A recent
Liver cirrhosis is known to cause an imbalance in the estrogen-anti-estrogen status of
146
CHANG & OHARA
lungsz1 and the absence of lung uptake of the body, in favor of the estrogens.' Mikhael 99n'Tc-sulfur colloids during liver-spleen and Evens36reported that estrogen treatment scans. In analogy to studies in experimental of guinea pigs and toads leads to mobilization animals, however, it is possible that PIMs of large numbers of phagocytic cells from may be inducible in humans under patholiver, spleen, and bone marrow into the intralogic conditions. In addition to one report vascular space. These cells then are trapped showing PIM-like cells in resected lung speciin the pulmonary microcirculation but retain men,16a series of case reports in nuclear medtheir ability to phagocytize colloidal particles. icine literature has suggested that pulmonary These authors suggest that increased levels of intravascular phagocytosis can occur in huestrogenic hormones stimulate the migration mans.23, 24, 27. 28 of reticuloendothelial cells to the lung and Dehring and Wismarlh studied electronmitherefore account for the occasional observasections of lung specimen resected tion of increased lung uptake of 9 9 m T ~ - ~ ~ l fcroscopic ur colloids in patients with liver disease.30 for various noninfectious indications. In 7 of 40 specimens, they were able to identify intraApplying our modern concept of the monovascular cells with morphology resembling nuclear phagocyte system to these results, PIMs in sheep, including the presence of their findings are consistent with estrogenphagocytic vacuoles and micropinocytosis induced stimulation of marrow-derived vermiformis. Junctional complexes between monocytes, with subsequent pulmonary sethese intravascular macrophages and endoquestration of the activated m o n ~ c y t e sIt . ~is~ thelial cells were not noted, however. The not clear whether prolonged stimulation with intravascular macrophages in these human estrogenic hormones would cause the activilung specimens were not quantitated, but ated monocytes to develop into intravascular they seemed less plentiful than the PIMs in macrophages. In addition to its potential effects on the Keyes et a124reported a series of 22 patients mononuclear phagocyte system, estrogen with marked increases in lung uptake of treatment has been shown to reduce hypoxic 9 9 m T ~ - ~ ~colloid l f u r during routine liver impulmonary vasoconstriction in the sheep aging. These cases represent 1.6% of all the lung.60Although this effect is small in magniliver scans performed in a 33-month period. tude, alterations in estrogen hormone levels In each case, technical factors that might lead in cirrhosis could contribute to the changes to increased lung uptake from microemboliin pulmonary vascular reactivity.1° zations were excluded. The most common diagnosis (found in ll patients) in this group Other Potential Mechanisms of patients was some form of malignancy. Of the 22 patients, 18 had some evidence of liver Because PIMs are derived from circulating abnormality, but only four had documented monocytes, cytokine factors that regulate the liver cirrhosis. It is noteworthy that the findgrowth and differentiation of monocytes and ing of marked lung uptake of ""Tc-sulfur monocyte precursor cells are likely to be imcolloid was associated with high mortality, portant in the induction of PIM in liver cirwith a median survival of only 3.5 months rhosis. One such factor is the macrophage after the liver scan.24 colony stimulating factor (CSF-l), which inIn a subsequent study, Klingensmith28reduces macrophage proliferation and differenported on 27 patients with increased lung t i a t i ~ n CSF-1 . ~ ~ content has been reported to uptake of 99mTc-sulfur colloid during routine be elevated in human liver disease.18Whether liver-spleen scans. A variety of disease proincreased activity of CSF-1 plays a role in the cesses existed in this group, including 12 cancellular alterations in the lung in rats with cers, six collagen vascular diseases, four biliary cirrhosis is unknown. Laennec's cirrhosis, and three infections. The survival in this group of patients was significantly better than in the group reported by PULMONARY INTRAVASCULAR Keyes et al.24Various other reports in the PHAGOCYTOSIS IN HUMANS nuclear medicine literaturez3,27, 52, 53 further support the association between increased It generally is accepted that normal human lung uptake of ""Tc-sulfur colloid and liver lungs do not contain PIMs. This belief is disease and malignancy, as well as other less based on lack of finding intravascular macrocommon disorder^.^ phages on morphometric studies of human
PULMONARY INTRAVASCULAR PHAGOCYTOSISIN LIVER DISEASE Table 2. CONDITIONS ASSOCIATED WITH PULMONARY INTRAVASCULAR PHAGOCYTOSIS Common Malignancy (breast carcinoma, lymphoma) Liver disease (cirrhosis, metastatic tumor, abscess) Infection (abscess) Uncommon Histiocytosis X Mucopolysaccharidosis type II Organ transplantation (bone marrow, liver) from Chang S: Pulmonary intravascular inflammation in sepsis and liver disease. Semin Respir Crit Care Med 15463, 1994; with permission.
The mechanism for the observed pulmonary intravascular phagocytosis in humans is unknown. Based on animal studies discussed earlier, we might speculate that the presence of intravascular macrophages or activated monocytes in the pulmonary microcirculation is responsible for the intense lung uptake of intravascular colloid particles. If so, this would indicate that PIMs could be induced in humans by some pathophysiologic stimuli associated with liver disease or malignancy. Much work remains to be done to clearly demonstrate the presence of PIMs in these settings and to elucidate the cellular and molecular mechanisms of the PIM induction. CLINICAL CONSEQUENCES OF PULMONARY INTRAVASCULAR PHAGOCYTOSIS Clearance of lntravascular Particles
Warner and Brain57 discussed potential functions of PIM in species (such as sheep and pigs) that have these cells in large numbers. In such animals, PIMs are an important part of the mononuclear phagocyte system and participate in the reticuloendothelial clearance of blood-borne particles. Using quantitative magnetometry, Brain et a157demonstrated that greater than 80% of injected iron oxide particles are removed from circulation by PIMs in the lungs of sheep, calves, and cats. PIMs also may help in removal of effete erythrocytes, given that erythrophagocytosis by PIMs has been reported by several 61
In addition to clearance of intravascular particles, PIMs also remove circulating bacteria from the blood stream, thereby participating in host defense against septicemia. Lungs containing PIMs also show increased uptake of circulating LPS, although cellular localiza-
147
tion of LPS within PIMs has not been demonstrated conclusively. Not unexpectedly, uptake of endotoxin by sheep lung is followed rapidly by inflammatory cell infiltration and signs of acute lung vascular injury.59 Pulmonary Hypertension
Although the incidence is relatively low, severe pulmonary hypertension is associated with a variety of chronic liver diseases in humans (discussed elsewhere in this issue). It is tempting to speculate that PIMs may play a role in the pathogenesis of this disorder because animals with PIMs are uniquely susceptible to severe pulmonary hypertension in response to intravascular particles and endotoxin. Miyamoto et a137showed that injections of liposomes in sheep cause dose-dependent rises in pulmonary arterial pressure mediated by thromboxane. The same response occurs with injection of monastral blue, a colloidal pigment.33In vitro, isolated PIMs from pig lungs metabolize arachidonic acid into various metabolites, including thromboxane B2.3 We also have documented increased thromboxane B, level in lung tissue from rats with biliary cirrhosis, and suggested that thromboxane A, may contribute to the increase in lung vascular permeability in these animals? In ex vivo perfused lungs from rats with biliary cirrhosis, we have found increased pulmonary hypertensive response to endotoxin and phorbol myristate acetate.56 The hypothesis that PIMs may play a role in the development of pulmonary hypertension will be very difficult to prove or disprove. The incidence of pulmonary hypertension in cirrhosis is low and there has not been any report of pulmonary intravascular phagocytosis occurring in the setting of pulmonary hypertension. Moreover, we have not noted any animals with pulmonary hypertension in our hemodynamic study of rats with biliary cirrhosis.1o Adult Respiratory Distress Syndrome in Liver Cirrhosis
Adult respiratory distress syndrome (ARDS) is characterized by acute lung vascular injury, increased vascular permeability, and noncardiogenic pulmonary edema. In 1987, Matuschak et a135reported that patients
148
CHANG & OHARA
with end-stage liver failure are highly susceptible to the development of sepsis-induced ARDS. Twenty-three (79%) of the 29 patients with end-stage liver failure requiring intensive care unit admission developed ARDS, whereas only 3 (6.8%) of 44 patients without liver failure developed ARDS. Once initiated, the ARDS was irreversible, with mortality of 100% in this series. ARDS also complicates the clinical course of patients undergoing liver transplantation. Takaoka et a154found 10 cases of ARDS in 198 patients undergoing orthotopic liver transplantation. The mortality in these patients with liver disease approached 8070, much higher than the 40% to 60% mortality quoted for ARDS patients in general. In a recent case report,% ARDS, occurring in the setting of liver allograft rejection, resolved promptly following hepatic retransplantation, suggesting that the presence of liver disease somehow contributes to the development and perpetuation of ARDS. Various theories have been proposed to explain the increased susceptibility of patients with chronic liver disease to ARDS (discussed elsewhere in this issue). Matuschak%,35 described four elements of the systemic inflammatory response that are under hepatic regulation: (1) clearance of endotoxin, bacteria, and vasoactive substances by hepatic Kupffer cells, (2) hepatic production of cytokine and eicosanoid mediators, (3) metabolic inactivation and detoxification of these mediators by Kupffer cell-hepatocyte interaction, and (4) hepatic synthesis of acute-phase protein, which moderates cytokine-mediated inflammation and tissue injury. It has been suggested that liver failure leads to the derangement of one or more of these mechanisms, thereby predisposing these patients to ARDS. Pulmonary lntravascular Phagocytosis and Adult Respiratory Distress Syndrome
The results of our recent study in rats with biliary cirrhosis suggest another potential mechanism to explain ARDS in patients with liver cirrhosis-the induction of PIMs. The comparative studies in different animal species have shown that sensitivity to endotoxin-induced lung injury correlates with the presence of intravascular macrophages. Animals with PIMs (sheep, pigs) are exquisitely sensitive to endotoxin, showing response to
microgram quantities, with severe pulmonary hypertension, increased vascular permeability, and lung edema. In contrast, animals without PIMs (rat, mice) can tolerate up to milligram quantities of endotoxin and, when they die of septic shock, their lungs usually are free of significant edema. In rats with biliary cirrhosis, we found both a quantitative and qualitative change in the pulmonary circulatory response to bacterial endotoxin" As the lungs acquired large numbers of intravascular macrophages, the rats became extremely endotoxin-sensitive.Shamoperated control rats tolerated up to 1 mg/ kg of endotoxin without any obvious adverse effects, but rats with biliary cirrhosis showed increased lethargy and piloerection with as little as 0.01 mg/kg of endotoxin. The median lethal dose for endotoxin was shifted by at least two log orders of magnitude in rats with biliary cirrhosis.8 Moreover, whereas control rats dying after high doses of endotoxin had normal lung weights, rats with biliary cirrhosis dying after low doses of endotoxin had markedly increased wet lung weights and lung wet-to-dry weight ratios, indicating the development of severe pulmonary edema? In another recent study, Kleber et alZ5 showed that endotoxin infusion causes marked reductions in cardiac index and renal blood flow in rats with biliary cirrhosis but not in sham-operated rats. Unfortunately, pulmonary circulatory effects of endotoxin were not measured in that study. Based on the results of these animal studies, as well as clinical reports of pulmonary intravascular phagocytosis in certain patients with chronic liver disease, we propose the following hypotheses: (1) liver cirrhosis induces the development of intravascular macrophages in the pulmonary circulation and (2) the presence of large numbers of PIMs predisposes these patients to develop ARDS following sepsis or endotoxemia. Certainly, additional clinical and experimental studies are necessary to confirm or refute these hypotheses. ARDS is a vexing clinical problem associated with high morbidity and mortality. Although certain predisposing medical conditions have been well described, ARDS occurs unpredictably among patients with common risk factors. It is clear that host factors are likely to be important in determining individual susceptibility to ARDS, yet none of these factors has been identified clearly. PIMs, by virtue of their unique location, can be stimu-
PULMONARY INTRAVASCULAR PHAGOCYTOSIS IN LIVER DISEASE
lated by circulating bacteria, endotoxin, or cytokines to release additional vasoactive mediators, such as tumor necrosis factor, eicosanoids, and reactive oxygen intermediates, directly into the pulmonary microcirculation. The resultant amplification of the systemic inflammatory response in the pulmonary microcirculation may lead to lung microvascular injury and the clinical syndrome of ARDS.
References 1. Adlercreutz H. Hepatic metabolism of estrogen in health and disease. N Engl J Med 290:1981, 1974 2. Bemelmans MHA, Gouma DJ, Greve JW, et a1 Cytokines tumor necrosis factor and interleukin-6 in experimental biliary obstruction in mice. Hepatology 15:1132, 1992 3. Bertram TA, Overby LH, Danilowicz R, et al: Pulmonary intravascular macrophages metabolize arachidonic acid in vitro. Am Rev Respir Dis 138:936, 1988 4. Bomzon A, Rosenberg M, Gali D, et al: Systemic hypotension and decreased pressor response in dogs with chronic bile duct ligation. Hepatology 6:595, 1986 5. Brigham KL, Meyrick B: Endotoxin and lung injury. Am Rev Respir Dis 133:913, 1986 6. Caramelo C, Femandez-Gallardo S, Santos JC, et al: Increased levels of platelet-activating factor in blood from patients with cirrhosis of the liver. Eur J Clin Invest 177,1987 7. Chane S: Pulmonarv intravascular inflammation in sepsizand liver disease. Semin Respir Crit Care Med 15463, 1994 8. Chang S, Ohara N: Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J Clin Invest 942009, 1994 9. Chang S, Ohara N Increased pulmonary vascular permeability in rats with biliary cirrhosis: Role of thromboxane A,. Am J Physiol264:L245, 1993 10. Chang S, Ohara N Pulmonary circulatory dysfunction in rats with biliary cirrhosis: An animal model of the hepatopulmonary syndrome. Am Rev Respir Dis 145:798, 1992 11. Chang S, Westcott JY, Pickett WC, et al: Endotoxininduced lung injury in rats: role of eicosanoids. J Appl Physiol 66:2407, 1989 12. Chang S, Feddersen CO, Henson PM, et al: Plateletactivating factor mediates hemodynamic changes and lung injury in endotoxin-treated rats. J Clin Invest 79:1498, 1987 13. Chida M, Spom PHS, Wamer AE, et a 1 Phagocytic activity of pulmonary intravascular macrophages from rats with biliary cirrhosis [abstract]. Am J Respir Crit Care Med 149:A1028, 1994 14. Chida M, Spom PHS, Wang X, et al: Perfusion isolation of pulmonary intravascular macrophages from rats with biliary cirrhosis [abstract]. Am Rev Respir Dis 147A307, 1993 15. Daoud FS, Reeves JT, Schaefer J W Failure of hypoxic pulmonary vasoconstriction in patients with liver cirrhosis. J Clin Invest 51:1076, 1972 16. Dehring DJ, Wismar BL Intravascular macrophages in pulmonary capillaries of humans. Am Rev Respir Dis 139:1027, 1989
149
17. Deviere J, Content J, Denys C, et al: Excessive in vitro bacterial lipopolysaccharide-induced production of monokines in cirrhosis. Hepatology 11:628, 1990 18. Esposti SD, Stanley R, Zem MA: Macrophage-colony stimulating factor (CSF-1) content is markedly increased in human liver disease [abstract]. Hepatology 12:908, 1990 19. Femandez-Munoz D, Caramelo C, Santos JC, et a1 Systemic and splanchnic hemodynamic disturbances in conscious rats with experimental liver cirrhosis without ascites. Am J Physiol 249:G316, 1985 20. Furukawa T, Yasumoto K, Inokuchi K Pulmonary interstitial edema in experimental cirrhosis of the liver in rats. Eur Surg Res 16:366, 1984 21. Gehr PM, Bachofen M, Weibel E R The normal human lung. Ultrastructure and morphometric estimation of diffusion capacity. Respir Physiol32121,1978 22. Gouma DJ, Coelho JCU, Fisher JD, et a1 Endotoxemia after relief of biliary obstruction by internal and external drainage in rats. Am J Surg 151:476, 1986 23. Imarisio JJ: Liver scan showing intense lung uptake in neoplasia and infection. J Nucl Med 16:188, 1975 24. Keyes JW Jr, Wilson GA, Quinones J D An evaluation of lung uptake of colloid during liver imaging. J Nucl Med 14:687, 1973 25. Kleber G, Braillon A, Gaudin C, et a1 Hemodynamic effects of endotoxin and platelet activating factor in cirrhotic rats. Gastroenterblogy 103:282, 1992 26. Klingensmith WC 111, Lovett VJ Jr: Lung uptake of "mTc-sulfur colloid secondary to intraperitoneal endotoxin. J Nucl Med 15:1028, 1974 27. Klingensmith WC 111, Ryerson TW, Corman JL: Lung uptake of 99mTc-sulfurcolloid in organ transplantation. J Nucl Med 14757,1973 28. Klingensmith WC 111, Yang SL, Wagner HN Jr: 99mTcsulfur colloid in liver and spleen imaging. J Nucl Med 19:31, 1978 29. Kountouras J, Billing BH, Scheuer PJ: Prolonged bile duct obstruction: A new experimental model for cirrhosis in the rat. Br J Exp Pathol 65305, 1984 30. Krowka MJ: Pulmonary aspects of chronic liver disease and liver transplantation. Mayo Clin Proc 60:407, 1985 31. Lee SS, Girod C, Braillon A, et al: Hemodynamic characterization of chronic bile duct-ligated rats: Effect of pentobarbital sodium. Am J Physiol 2 5 ~ ~ 1 7i986 6, I1 stimulates earlv 32. Liu F-Y. Coean MG: Aneiotensin " proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate. J Clin Invest 84:83, 1989 33. Longworth KE, Westgate AM, Grady MK, et al: Development of pulmonary intravascular macrophage function in newborn lambs. J Appl Physiol 73:2608, 1992 34. Matuschak GM, Shaw BW: Adult respiratory distress syndrome associated with acute liver allograft rejection: Resolution following hepatic retransplantation. Crit Care Med 15:878, 1987 35. Matuschak GM, Rinaldo JE, Pinsky MR, et al: Effect of end-stage liver failure on the incidence and resolution of the adult respiratory distress syndrome. J Crit Care 2162, 1987 36. Mikhael MA, Evens RG: Migration and embolization of macrophages to the lung: A possible mechanism for colloidal uptake in the lung during liver scanning. J Nucl Med 1622, 1975 37. Miyamoto K, Schultz E, Heath T, et al: Pulmonary intravascular macrophages and hemodynamic effects of liposomes in sheep. J Appl Physiol 641143, 1988 I
"
150
CHANG&OHARA
38. Murray BM, Paller M S Decreased pressor reactivity to angiotensin I1 in cirrhotic rats. Evidence of a postreceptor defect in angiotensin action. Circ Res 57424,1985 39. Naeije R, Hallemans R, Mols P, et a 1 Hypoxic pulmonary vasoconstriction in liver cirrhosis. Chest 80570, 1981 40. Newman JH, Loyd JE, English K, et al: Effect of 100% oxygen on lung vascular function in awake sheep. J Appl Physiol M1379, 1983 41. Noda T, Mimura H, Orita K Assessment of Kupffer cell function in rats with chronic liver injury caused by CCb. Hepatogastroenterology 37319, 1990 42. Ohara N, Voelkel NF, Chang S Tissue eicosanoids and vascular permeability in rats with chronic biliary obstruction. Hepatology 18:111, 1993 43. Ohgami M, Doerschuk CM, Gie RP, et a1 Late effect of endotoxin on the accumulation and function of monocytes in rabbit lungs. Am Rev Respir Dis 146:190, 1992 44. Paumgartner G, Longueville J, Leevy CM: Phagocytic activity in experimental liver injury. Exp Mol Pathol 9:161, 1968 45. Prytz H, Jolst-Christensen J, Korner B, et al: Portal venous and systemic endotoxemia in patients with cirrhosis. Scand J Gastroenterol 112357, 1976 46. Quinones JD. Localization by technetium-sulfur colloid after RES stimulation. J Nucl Med 1 4 4 3 , 1973 47. Rodman T, Sobel M, Close HP: Arterial oxygen unsaturation and the ventilation-perfusion defect of Laennec’s cirrhosis. N Engl J Med 263:73, 1960 48. Rodriquez-Roisin R, Roca J, Agusti AG, et al: Gas exchange and pulmonary vascular reactivity in patients with liver cirrhosis. Am Rev Respir Dis 135:1085, 1987 49. Saha GB, Feiglin DHI, O’Donnell JK, et al: Experience with 99mTechnetiumalbumin colloid kit for reticuloendothelial imaging. J Nucl Med Techno1 14149,1986 50. Sherr CJ: Regulation of mononuclear phagocyte pro-
51. 52. 53. 54. 55. 56.
57. 58.
59. 60. 61. 62.
liferation by colony-stimulating factor-1. Int J Cell Cloning 8(suppl 1):46, 1990 Smith JB, Lincoln TM: Angiotensin decreases cyclic GMP accumulation produced by atrial natriuretic factor. Am J Physiol 253C147, 1987 Stadalnik RC: Diffuse lung uptake of Tc-99m-sulfur colloid. Semin Nucl Med 10:106, 1980 Steinbach H L Pulmonary accumulation of 9ymTc-sulfur colloid during liver scanning. Texas Med 68:137, 1972 Takaoka F, Borwn MR, Paulsen AW, et al: Adult respiratory distress syndrome following orthotopic liver transplantation. Clin Transplantation 3:294,1989 Voelkel NF: Mechanisms of hypoxic pulmonary vasoconstriction. Am Rev Respir Dis 133:1186,1986 Wang X, Ohara N, Chang S Enhanced pulmonary hypertensive responses to endotoxin and phorbol myristate acetate in rats with chronic biliary obstruction [abstract]. Clin Res 40697A, 1992 Warner AE, Brain JD: The cell biology and pathogenic role of pulmonary intravascular macrophages. Am J Physiol 258L1, 1990 Warner AE, =amp MM Jr, Bellows CF, et al: Endotoxemia enhances lung uptake of circulating particles in species lacking pulmonary intravascular macrophages [abstract]. Am Rev Respir Dis 139:A158, 1989 Warner AE, Decamp MM Jr, Molina RM, et a1 Pulmonary removal of circulating endotoxin results in acute lung injury in sheep. Lab Invest 59:219, 1988 Wetzel RC, Zacur HA, Sylvester JT: Effect of puberty and estradiol on hypoxic vasomotor response in isolated sheep lungs. J Appl Physiol 56:1199, 1984 Winkler GC, Cheville NF: Monocytic origin and postnatal mitosis of intravascular macrophages in the porcine lung. J Leukocyte Biol 38:471, 1985 Yoshimura K, Todd ML, Pier KG, et a1 Effects of thromboxane A, analogue and prostacyclin on lung fluid balance in newborn lambs. Circ Res 65:1409, 1989
Address reprint requests to Shih-Wen Chang, MD Midwest Consultants of Internal Medicine 2640 Milton Avenue PO Box 505 Janesville, WI 53547-0505