Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115
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ABSTRACT |
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Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria. Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent. Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection. PFCE pretreatment decreased the rate and extent of vascular clearance of P. aeruginosa, with decreased uptake by the liver. Importantly, there were significant decreases in killing of P. aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals. PFCE did not alter the clearance of S. aureus from the circulation. However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake. Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization. These experiments demonstrate that the clearance kinetics and organ distribution of circulating P. aeruginosa and their subsequent killing are altered by PFCE. Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P. aeruginosa.
lung injury; liver; macrophage
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INTRODUCTION |
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BLOOD SUBSTITUTES have been developed for various reasons. Patients who are Jehovah's Witnesses refuse to accept human blood because of their religious beliefs. In some emergencies, adequate supplies of human blood may be lacking. Finally, there are persistent concerns about viral contamination (e.g., hepatitis and human immunodeficiency virus-1) of human blood products. For almost two decades, perfluorochemical emulsions (PFCE) have been studied as alternatives to blood in both animals and humans (10, 13, 14).
Although clinical trials have been conducted to evaluate the safety and efficacy of PFCE for use in patients (22, 25), the effects of PFCE on the organ systems that they perfuse have not been well studied. A number of investigators have raised concerns about the fate of the intravenously injected PFCE. Some of the PFCE is cleared from the blood by phagocytic cells that have access to the circulating blood (the mononuclear phagocyte system, MPS). Several papers have described interactions of PFCE with the MPS (1, 3, 8, 20, 26). There are indications that there can be depression in their phagocytic function.
We have previously demonstrated significant changes in the ultrastructure of rat Kupffer cells after intravenous injection of PFCE (33). These hepatic macrophages contained abundant vacuoles, many apparently filled with particles of PFCE. Associated with these morphological changes was a reduction in the motion of particle-containing phagosomes and phagolysosomes (1, 33) as detected with magnetometric methods (4, 11). We also observed decreased rates of clearance of inert insoluble 57Co3O4 particles from the blood and altered organ distribution of these particles in PFCE-injected rats (6). We found that PFCE administration decreased liver uptake, with compensatory increases of cobalt oxide particle uptake in the lungs and spleen.
The primary goal of this study was to examine the effects of PFCE on the function of the components of the MPS with access to the circulating blood in regard to the fate of circulating bacteria. Our goal was to evaluate the clearance kinetics, organ distribution, and intracellular bacterial killing in control rats and in those given PFCE.
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats (mean weight of 243 ± 8 g) were purchased from Charles River Laboratories (Wilmington, MA). All animals were allowed to acclimate for 1 wk after arrival and were given free access to food and water. One group of rats was preinjected with three daily tail vein injections of PFCE at 2 ml/100 g body wt. This protocol was chosen on the basis of a calculated blood volume addition of ~25%. Age- and weight-matched controls were similarly injected with physiological saline. The lungs and other major organs were grossly and histologically free of pneumonia and parasites postmortem.
Blood substitute (PFCE). The PFCE was prepared by sonicating the components in a CO2 atmosphere to prevent fluoride ion production (12). The emulsion contained 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane (Suntech, Marcus Hook, PA). Pluronic F-68 (BASF Wyandotte Chemical, Wyandotte, MI) was used as the emulsifying agent at a final concentration of 2.5% (wt/vol). Hydroxyethyl starch (3%, wt/vol, in the final emulsion) served as the oncotic agent. The aqueous phase of the PFCE was a modified Ringer bicarbonate solution. Osmolarity of the PFCE was 290 ± 2 mosmol/l, and the pH was adjusted to 7.45. The emulsion was filtered through a 0.45-µm cellulose acetate membrane filter (Millipore, Bedford, MA) and was stored at 4°C until needed. Perfluorochemical particle diameters in the emulsion ranged from 0.05 to 0.4 µm, with an average of ~0.2 µm as estimated by electron microscopy. Filtration of the emulsion through the cellulose acetate filter removed any perfluorochemical particles larger than 0.45 µm. The endotoxin contamination of the preparation was indirectly evaluated by a pyrogen test, and all preparations were certified to be pyrogen free.
Bacteria. Pseudomonas aeruginosa (strain P220; courtesy Dr. James Pennington) growing exponentially in trypticase soy broth were washed two times in sterile saline and resuspended at an optical density (620 nm) of 1.2 in sterile saline. Staphylococcus aureus (courtesy of Dr. Richard Rose) were similarly grown and washed and suspended in sterile saline to an optical density (510 nm) of 1.2. The S. aureus was a clinical isolate from a blood culture from a diabetic patient at the Beth Israel Deaconess Hospital (Boston, MA). When radiolabeled, S. aureus were inoculated into trypticase soy broth with 10 ml of 59Fe citrate (1.0 mCi/ml; New England Nuclear) and incubated at 37°C in a shaker bath overnight.
Administration of bacteria. The bacterial suspension was injected at a mean volume dose of 0.33 ml/100 g body wt [mean doses of 20 × 107 colony-forming units (CFU)/100 g for P. aeruginosa and 7 × 107 CFU/100 g for S. aureus]. These doses were both sublethal and comparable to those used in previous studies involving bacterial injection in small mammals (2). The rats were anesthetized with vaporized halothane to allow catheterization of a jugular vein and serial collection of blood samples for 1 h. Single 20-µl samples were withdrawn via the jugular catheter both before and at 1, 3, 5, 10, 15, 20, 30, and 60 min after injection to determine the kinetics of bacterial clearance. Serial 10-fold dilutions of blood were made in distilled water so that cells, but not bacteria, were lysed. Next, 1-ml aliquots of each were plated in trypticase soy agar using standard pour-plate techniques.
Analysis of bacterial content. Rats used for this analysis were briefly anesthetized with vaporized halothane and were injected intravenously with the bacterial suspension. At 1 and 4 h postinjection, the rats were humanely killed. The thorax and abdomen were aseptically opened; next, the liver, spleen, kidneys, and right lungs were removed. The left lung was fixed for examination by light and electron microscopy. The liver, spleen, kidneys, and right lung were weighed, and one to four randomly chosen samples (0.5-1.0 g) were selected from each organ. The samples were weighed, and homogenates were prepared in a tissue grinder using sterile distilled water. Serial 10-fold dilutions of each homogenate were made in sterile distilled water, and 1-ml aliquots were plated in trypticase soy agar.
The number of colonies on each plate was counted after a 24-h incubation at 37°C using a Quebec colony counter; the colony-forming units per gram of tissue for each organ and the colony-forming units per milliliter of blood for each time point were determined. Total organ weights were used to calculate the colony-forming units in each organ. The total recovery at 1 and 4 h, the percentage of the recovered dose localized in each organ, and the number of organisms in the circulating blood at each time point were computed (18). When radioactive bacteria were used, weighed samples of tissues and aliquots of blood were placed in test tubes and assayed for 59Fe content in a Packard model 2001 gamma counter. In a manner similar to that described above, the organ distribution of the label was computed as the percent of total recovered radioactivity. Additional rats were intravenously injected with 5 to 10 times more bacteria to facilitate ultrastructural demonstration of cells responsible for organ uptake. The organ distribution of recovered bacteria at this higher dose was approximately similar to that observed with lower doses (unpublished observations).
Bacterial killing assay. The ability of phagocytic cells in different organs to kill retained bacteria was evaluated by comparing the number of colony-forming units recovered from the various organs at 1 h (initial uptake) with the number found 3 h later (4 h after injection). Bacterial killing in each organ was calculated as the percent change in colony-forming units per gram from 1 to 4 h postinjection as follows
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Histological and ultrastructural methods. Before death, each rat was anesthetized with an intraperitoneal injection of pentobarbital sodium, and its trachea was cannulated. The abdominal cavity was opened, both hemidiaphragms were punctured, and 2% glutaraldehyde in 0.084 M sodium cacodylate buffer (pH 7.4) entered the lungs via the trachea at a constant pressure of 20 cmH2O. Tissue samples for electron microscopy were cut from transverse slices through the apical and basal regions of the left lung of each rat. These samples were washed in cacodylate buffer, postfixed in 2% osmium tetroxide, dehydrated in a series of alcohol and propylene oxide, and embedded in epoxy resin. Samples were randomly selected from the pool of blocks for each animal; thin sections were cut from these on a Sorvall MT 6000 ultramicrotome using a diamond knife, picked up on 200-mesh uncoated grids, and then stained with uranyl acetate and lead citrate. Electron micrographs were taken on a Philips 300 electron microscope. Sections 0.5-1.0 µm in thickness were cut with glass knives and stained with toluidine blue for light microscopic examination.
Statistical analyses. Student's t-test was used for comparison of means. Statistical significance was assumed at P < 0.05.
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RESULTS |
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Kinetics of vascular clearance of injected
bacteria. The rates of disappearance of
P. aeruginosa and S. aureus from the blood during the first hour after their
intravenous injection are shown in Fig. 1.
The rate of clearance of P. aeruginosa
was considerably slowed by PFCE pretreatment (Fig.
1A). At all time points between 10 and 60 min, there was a significant difference between the amount of
bacteria in the blood when the PFCE group was compared with the saline
group (P < 0.05). One hour after
injection of bacteria, nearly 100 times more bacteria remained in the
blood of PFCE-treated rats than in control rats. In contrast, no
significant difference in the clearance kinetics of S. aureus between the two groups of animals was observed
(Fig. 1B). We also utilized a
radioactive label to follow S. aureus
clearance (data not shown). We found that the clearance half-time for
the iron label appeared longer in PFCE-treated rats than in the
corresponding controls (2.16 ± 0.23 vs. 1.67 ± 0.34 min,
although P > 0.05). At times after
15 min, there was more radioactivity measured in the blood than would
be predicted by the colony-forming unit counts. This may reflect a more
rapid liberation of 59Fe from the
ingested bacteria in the control rats.
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Organ distribution of recovered
bacteria. The distribution of recovered bacteria
throughout the body is summarized in Fig. 2. For both bacteria used, the hepatic
uptake was significantly diminished by PFCE. For P. aeruginosa (Fig.
2A), the liver content decreased
from 87.96 ± 5.48 to 68.96 ± 5.19% of the recovered dose. For
S. aureus (Fig.
2B), the liver uptake decreased from 93.79 ± 1.2 to 81.74 ± 3.86%. Also consistent with Fig.
1A, the animals injected with
Pseudomonas had significantly more
viable organisms in the blood at the time of death. In the animals
given S. aureus, the reduction in
hepatic uptake was accompanied by significant increases in lung and
kidney uptake. We observed alterations in the distribution of the
59Fe label of the
S. aureus that were consistent with
Fig. 2B. A significant decrease was
seen in hepatic content, and significant increases were also seen in
the pulmonary and renal contribution (data not shown). The percent of
injected dose of P. aeruginosa recovered 1 h after intravenous injection was significantly higher in
PFCE-injected rats than in saline control rats. For P. aeruginosa, 47.0 ± 14.0% of the injected
dose was recovered in the PFCE-injected rats compared with only 14.4 ± 4.7% in saline controls. For S. aureus, the values were 35.4 ± 17.7 and 52.3 ± 14.4 %, respectively.
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Bacterial killing. When we examined
the bactericidal activity of the major organs that retained the
injected P. aeruginosa, we observed a
dramatic change induced by pretreatment with the artificial blood. PFCE
caused a significant decrease in the microbicidal activity of all four
of the organs studied (P < 0.05).
The changes in the number of viable P. aeruginosa between 1 and 4 h (the times of death for
the two cohorts) are shown in Fig.
3A. In the
liver, spleen, lungs, and kidneys, the saline control animals were able to kill between 77 and 94% of the retained bacteria. In marked contrast, bacterial killing was significantly decreased in
PFCE-injected rats. Killing in the spleen was reduced from 79 to 16%
during the 3-h observation period. The bactericidal activity in the
liver, lungs, and kidneys was impaired to an even greater extent. In all of these three organs, significant growth of P. aeruginosa was observed. The number of colony-forming
units increased by 58% in the liver, 136% in the lung, and 49% in
the kidneys. However, no significant change in the killing of
S. aureus was observed in
PFCE-injected rats. As shown in Fig.
3B, both PFCE-treated and control
animals eliminated the majority of ingested S. aureus in all of the organs examined.
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Morphological changes caused by PFCE.
The three daily intravenous injections of PFCE caused profound
morphological changes that were evident at postmortem examination. The
body weights of PFCE-injected rats were not different from those of the
saline control. However, the increase in body weight over the 3-day
period of the PFCE-injected rats was significantly lower than that in saline-injected controls (2.99 vs. 6.15% weight increase). The liver
and spleen weights from the PFCE-injected animals expressed as percent
of the whole body weight were higher than in saline controls (Table
1). The liver-to-body weight ratio was 13%
higher in PFCE than in control animals, and the spleen-to-body weight ratio doubled. Also evident was the pallor of the organs from the
PFCE-injected rats compared with that in the saline controls (Fig.
4).
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Microscopic examination also revealed morphological changes in the
liver induced by PFCE. As shown in Fig. 5,
A and
B, the liver was dramatically altered.
Although no gross necrosis or inflammation was evident, the most
striking change was the large number of foamy-appearing cells. Both
Kupffer cells and hepatocytes contained large numbers of vacuoles,
which probably represent ingested PFCE. Electron microscopy revealed
ultrastructural changes consistent with these observations.
Accumulation of PFCE within Kupffer cells was frequently observed.
Hepatocytes and endothelial cells appeared relatively normal but had
occasional clusters of PFCE-containing vacuoles. Kupffer cells, in
contrast, were often distorted by large complex vacuoles of PFCE (Fig.
5B). As shown in Fig.
5B, the hepatocytes appear normal
except for occasional clusters of PFCE-containing vacuoles. The overall
appearance of the pulmonary parenchyma was relatively normal (Fig.
5C). There was no evidence for an
inflammatory response within alveolar spaces. Nevertheless, cells
filled with intracellular PFCE particles were frequently seen. At the
light microscopy level, it was difficult to distinguish intravascular
monocytes from connective tissue macrophages. Occasionally, even
alveolar macrophages with similar vacuoles were seen. Electron
microscopy revealed the presence of PFCE inclusions within endothelial
cells and marginated monocytes (Fig.
5D).
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Cellular uptake of bacteria.
Ultrastructural examination of the lungs and liver revealed that
phagocytic cells were primarily responsible for removing bacteria from
the circulating blood. The characteristic appearance of
P. aeruginosa in the liver and lungs 1 h after injection is shown in Fig. 6,
A and
B. Hepatic macrophages containing PFCE
vacuoles were capable of ingesting P. aeruginosa (Fig.
6A). A quantitative assessment of
the site of P. aeruginosa uptake in
the liver from PFCE-treated animals revealed that 55% were within
Kupffer cells, 2% were in polymorphonuclear cells (PMN), and 43% were
in the capillary lumen either free in plasma or adherent to sinusoidal
cells.
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In the lungs, P. aeruginosa were seen far less frequently as is consistent with Fig. 2A. However, free bacteria were occasionally seen in the blood, an observation consistent with Fig. 1A, showing increased persistence of P. aeruginosa in the circulating blood. When ingested bacteria were seen, they were always seen within marginated phagocytic cells, usually monocytes. For example, Fig. 6B shows a large marginated mononuclear cell containing a bacterium.
The appearance of S. aureus in the
liver and lungs 1 h postinjection is shown in Fig.
7. The findings are similar to those seen
with P. aeruginosa. Intracellular
bacteria were confined to phagocytic cells. In the liver, 85% of
localized bacteria were found within Kupffer cells, with the remaining
15% found within PMN (Fig. 7A).
Many of these cells also contained small and large vacuoles
characteristic of PFCE-injected animals. In the lungs, we also observed
participation of both PMN as well as circulating monocytes in bacterial
localization (Fig. 7B). Many PMN
were seen containing ingested bacteria. Generally, PMN had fewer PFCE
vacuoles than did the monocytes.
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DISCUSSION |
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Side effects of PFCE. Our data show that injection of the PFCE in the blood may have a major impact on phagocytic cells, which have access to the circulating blood. This is consistent with several previous studies in vitro of commercially available PFCE (7, 17, 19). Our data clearly demonstrate that both circulating white blood cells as well as resident macrophages in such organs as the lungs and liver soon contain significant amounts of PFCE vacuoles. Particularly in mononuclear phagocytes, this results in the appearance of large vacuoles and compromised organelle motion. After injections of magnetic iron oxide particles and PFCE into rats, Weinstock et al. (33) used noninvasive magnetometric methods to study the effects of PFCE on phagocytic cells in the liver. The authors reported that magnetic relaxation, a measure of the spontaneous motion of phagosomes within cells, decreased in the livers of animals that received PFCE compared with those that received saline. Weinstock et al. also noted that the majority of iron particles and PFCE particles were within Kupffer cells. A similar decrease in phagosomal motion in Kupffer cells after PFCE and other lipid emulsion administration was reported later (1, 21).
These morphological and magnetometric changes induced by PFCE administration were also accompanied by functional changes. We have reported that PFCE are associated with decreased hepatic and increased pulmonary uptake of injected cobalt oxide particles (6). In the present study, P. aeruginosa were cleared more slowly from the blood, so that 100 times more viable bacteria were still present in the blood at 1 h compared with those in saline control animals. The organ distribution of retained viable organisms was also significantly altered. The hepatic contribution to vascular clearance was significantly decreased, resulting in the persistence of P. aeruginosa in the circulation. Because bactericidal activity of the liver was significantly decreased, that also increased the bacterial burden of other reticuloendothelial organs, including the lungs. Importantly, there was significant bacterial growth in the lungs, kidneys, and liver of PFCE-injected rats during a 3-h period.
Surprisingly, in spite of these major changes in the way P. aeruginosa, a gram-negative bacteria, was handled, the kinetics of S. aureus (gram-positive) clearance and killing was essentially unchanged. This may reflect different mechanisms of bactericidal activity, such as difference in the relative role of PMN versus mononuclear cells. It could also reflect the slightly lower dose of S. aureus that was used in this study (7 × 107 CFU/100 g vs. 20 × 107 CFU/100 g). Perhaps there could be an impairment in the clearance and killing of S. aureus at higher injected doses of either PFCE or bacteria. Nevertheless, PFCE treatment with this protocol did alter the distribution of cleared S. aureus away from the major reticuloendothelial organ, the liver, shifting bacterial uptake to other organs such as the kidneys, spleen, and lungs.
What are some of the possible mechanisms that explain how PFCE administration changes the clearance, organ distribution, and killing of intravenously injected P. aeruginosa? There are at least four possible mechanisms. First, the phagocytic cells are clearly filled with perfluorochemical, and thus they may be "overloaded." Previous phagocytic activity devoted to PFCE ingestion may have reduced the available number of bacterial receptors on the plasma membrane. In addition, the presence of PFCE may be a mechanical obstruction to organelle motion, ingestion, and killing. Intracellular killing involves careful orchestration of organelle motion. For example, phagosomes must be formed from plasma membrane, brought deep inside the cell; next, phagosome-lysosome fusion must take place. Second, these functional changes may reflect damage to mononuclear cells. Many have large vacuoles with no visible internal structure. Rather than being simply coalesced ingested PFCE droplets, these large vacuoles may also reflect cellular damage. Third, there may be fibronectin depletion (23). This protein is important for reticuloendothelial system activity. Because Kupffer cells take up abundant PFCE droplets, the circulating fibronectin levels may be reduced. Fourth, hepatic bacterial clearance is also related to hepatic blood flow. Mechanical obstruction as well as cytokine responses might be involved. It is possible that blood flow to the liver is reduced because of the enlarged PFCE-loaded Kupffer cells, leading to the observed enlargement of the liver after multiple PFCE injections. Likewise, PFCE may cause injury and swelling of the endothelium. This probable increased vascular resistance in the liver might contribute to the longer rates of clearance and altered organ distribution.
Mechanisms and importance of pulmonary uptake. As noted earlier, reduced hepatic uptake was accompanied by compensatory increased uptake in other organs such as spleen, kidneys, and lungs. The dose of bacteria to which the lungs are subjected is an important determinant of lung injury. Previous work in this laboratory (5, 28-30, 32) has emphasized the importance of pulmonary intravascular macrophages (PIMs). Present in ruminants, as well as in other animals such as pigs, horses, and cats, these resident cells in lung capillaries avidly phagocytize both test particles such as gold colloid and iron oxide (27, 28) as well as more relevant materials such as P. aeruginosa (32). Could such cells be responsible for the increased pulmonary uptake observed here? In a comparative study of 13 animals (5), pulmonary uptake of tracer particles is minimal in normal rats compared with that in PIM-containing species such as sheep, calves, pigs, goats, and cats. Those data are consistent with the values obtained for control rats seen in this study. Moreover, extensive electron microscopic studies failed to show any "classic" PIMs in rats. Mature macrophages adherent to the underlying endothelium with characteristic junctional complexes were not observed in rats (27, 34). These junctional complexes involve the plasma membranes of the macrophage and the underlying endothelial cell coming within 10 nm of each other; electron-dense material can also be seen enveloping both plasma membranes. No such structures were seen in either the control rat lungs or those treated with PFCE.
However, there are circumstances, like PFCE administration, when margination of leukocytes in lung capillaries may increase. There may even be circumstances in which PIMs can develop in non-PIM species (9, 31). Yet, little evidence for it was seen in this study. Instead, the bacteria were always contained in what appeared to be marginated mononuclear cells (probably circulating monocytes) as well as in PMN. These cells not only contained the bacteria but were also loaded with PFCE droplets in varying amounts. We do not know whether PFCE increased the likelihood of these cells marginating in the lung. Increases in cell size or stiffness could contribute. Alternatively, Kupffer cell failure might have led to increased ingestion of bacteria by circulating white blood cells. Next, even normal numbers of marginated white blood cells could give rise to the increased lung burdens of bacteria we observed. Labeling of white blood cells with quantitative measurements of the marginated pool in the lungs and other organs would be necessary to distinguish among these various possibilities.
Liver-lung relationships. Our data add weight to the hypothesis that diminished hepatic function can lead to increased pulmonary pathogen burden and may thus predispose to respiratory failure. This possibility has been raised in a slightly different context by a number of investigators such as Saba (23), who emphasized the possibility that excessive Kupffer cell activity would lead to fibronectin depletion and thus reduced phagocytic performance by Kupffer cells. In turn, this would lead to increased persistence of various pathogenic materials in the blood and thus increase the risk of pulmonary uptake and ultimately lung injury and adult respiratory distress syndrome. Our data do suggest that Kupffer cell function can modulate pulmonary burdens. The data presented in this paper provide a compelling example. We observed reduced hepatic uptake with concomitant increases elsewhere. There is little evidence to suggest that normal humans have significant pulmonary uptake of circulating test particles or pathogens. However, there are numerous reports suggesting that, with certain liver abnormalities, there is significant pulmonary localization of technetium sulfur colloid during liver scintigraphy (15, 16, 24). The data presented here also suggest that liver failure, especially of the Kupffer cells, could lead to increased pulmonary pathogen burden and thus enhance the probability of pulmonary infection and injury.
We showed that multiple PFCE intravenous administrations result in reduced capacity of the reticuloendothelial system to eliminate injected gram-negative bacteria from the blood. Uptake of bacteria by the liver is diminished in PFCE-injected rats, with compensatory increases in uptake by the lungs, kidneys, and spleen. In the liver, the principal cells responsible for uptake of injected bacteria are the Kupffer cells. In the lungs, marginated monocytes and PMN ingest the bacteria. Exposure to PFCE produces major changes in the ability of phagocytic cells to kill bacteria they have ingested. In fact, we observed P. aeruginosa proliferation in liver, kidney, and especially in the lungs of PFCE-injected rats. Our evidence suggests that the use of this perfluorochemical preparation as a blood substitute may alter host defenses and may place the patient at increased risk when bacterial infection is present.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. D. Brain, Dept. of Environmental Health, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
Received 7 November 1997; accepted in final form 24 February 1999.
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