THE EFFECT OF ENDOTOXIN O N IN VlVO RAT ALVEOLAR MACROPHAGE PHAGOCYTOSIS

Charles W. Frevert LllnJJachusettc, L S A , and D2ii)iori of Pulmonay and Ciitical Cate .Zledicine, Cnir Mia 5 h ing ton, Seat t le. Lt'a 5 hi ng /on. C Srl

Angeline E. Warner BoJton, 3 l a 1 wrhusettJ. C-J.4, and A RC, Harrard AledzLal S l h o o l , Boston, A \ l n s ~ a i h u ~ e t t ~ , C SA

Edie Weller A21aJJarhucett t . I 5.4

Joseph D. Brain .\la> tnthiitetf , . 1 -S4

0 Phjsiology Progiam, H a t r a i d School of Publit Heal th , BoJton,

0 Ph,stologl Piogram, H a i r a i d School oj Publzt Heal th ,

Department nf R i o ~ t a t z ~ t ~ c s , H a t z a t d Jthool of Public Heal th , Boston,

0 PhjJiolog) Ptoqtam. Harraid School of Public Henl th , Ro~ton,

Bacterial pneumonia is often associated with adult respiratory distress syn- drome (ARDS) [ 1-51, intra-abdominal infections [S], and bacteremia [7]. Because endotoxemia is a common occurrence in ARDS [ 8 ] , intra-abdominal infections [9], and bacteremia [lo], we wanted to know whether endotoxin,

Received 25 July 1995 ; accepted 20 May 1998. Supported in part by NIH grants Pol-HL-43510 and HL02374. Address correspondence to Charles W. Frevert, DVM, ScD, Seattle VAMC Pulmonary Research

Group, Seattle VA Medical Center, 151L, 1660 S. Columbian Way, Seattle, WA 98108, USA. E-mail: cfrevert@u.washington.edu

Experimental Lung Research, 24: 745-758, 1998 Copyright 1998 Taylor & Francis

0190-2148/98 812.00 + .OO 745

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7 46 C. W. Frevert et al.

a component of the cell wall of gram-negative bacteria, would impair alveol- ar macrophage (AM) function in an animal model.

Early studies of pulmonary host defenses showed that AM played an important role in the elimination of bacteria from the lungs [l 11. At 4 hours post bacterial inoculation, AM products recruit polymorphonuclear neutro- phils (PMN) into the lungs [12]. When AM-depleted animals were studied for 3 days, there was 100% lethality versus 100% long-term survival in the control group [13].

The effects of endotoxin on in vitro AM function have been extensively studied [ 14-1 71. Several investigators have suggested that AM phagocytosis in vivo is decreased in animals with endotoxemia [16, 18, 191. Decreased AM phagocytosis could impair pulmonary bacterial clearance and predispose the lungs to infection.

The purpose of this study was to investigate the effect of endotoxin on in vivo AM phagocytosis of radioactive gold colloid ( 1 9 8 A ~ ) in the lungs. Endo- toxin was administered either intratracheally ( IT) or intravenously (IV) to rats before in vivo AM phagocytosis was measured. We also studied the ability of entotoxin to influence cell populations and albumin levels in bron- choalveolar lavage (BAL) fluid in these animals.

MATERIALS AND METHODS

Experimental Design

Virus antigen-free male CD rats (Charles River, Kingston, NY), weigh- ing 250-350 grams, were used in this study. IV and I T procedures were performed while the rats were anesthetized with halothane (Halocarbon Labs, North Augusta, SC). IV administration was performed by tail vein injection with a 25-gauge butterfly catheter (Abbott Laboratories, Chicago, IL). The larynx was visualized directly for transpharyngeal IT adminis- tration with transillumination while the anesthetized animal was held in a vertical position. An 18-gauge blunt ended needle (Popper and Sons Inc., New Hyde Park, NY), 3 inches long, was used for I T installations.

For IV administration, lipopolysaccharide (LPS) was diluted in pyrogen- free saline and given at 0, 1, 2.5, and 5 mg/kg, in a volume of 0.33 mL per 100 grams body weight (BWT). For I T instillation, LPS was diluted in pyrogen-free saline and given at 0, 1, 5, and 10 mg/kg, in a volume of 0.15 mL per 100 grams BWT. The LPS was Escherichia coli serotype 01 11 : B4 (Sigma Chemical, St. Louis, MO). After LPS administration at 0 hour, the animals were followed for 22.5 hours and then given I9*Au I T while under halothane anesthesia. Colloidal particles (mean particle diameter of 30 nm, range of 5-50 nm, Amersham Corporation, Chicago, IL) of 1 9 8 A ~ were diluted in pyrogen-free saline and instilled in a volume of 0.15 mL per 100

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Endotoxin Enhances Macrophage PhagocytosiJ 747

grams BWT. Ninety minutes post-Au instillation and 24 hours after LPS administration, the animals were anesthetized by intraperitoneal injection with pentobarbital sodium (50 mg/kg, Anthony Products CO, Arcadia, CA 91006) and exsanguinated by cutting the abdominal aorta.

Bronchoalveolar Lavage

To prepare the animals for BAL, the lungs were collapsed by making a small hole in the diaphragm through an abdominal incision. While main- taining an intact thorax, the cervical trachea was exposed and cannulated with polyethylene tubing (PE190). BAL was performed by doing 12 3-mL washes of the lungs with physiologic saline (0.9Y0 NaCI). Gentle massage of the thorax (1 minute per lavage) was done to maximize cell recovery. Fluid recovered from lavages 1 and 2 were kept separate; the remaining lavages were paired (3-4, 5-6, 7-8, 9-10, and 11-12) and placed on ice.

Cell Enumeration

The number and identity of cells for lavages 1 and 2 as well as each paired lavages were determined. Macrophage numbers were obtained by counting cells in a hemocytometer at a magnification of 100 x ; cell size and the granularity of the cytoplasm were used to differentiate macrophages from lymphocytes and neutrophils (PMN). Differential cell counts were performed by counting 200 cells (magnification of x 1000) on cytospin slides stained with Diff-Quik stain (American Scientific Products, McGaw Park, IL). PMN numbers were calculated by comparing the PMN to macrophage ratios from the differential cell counts to total macrophage numbers.

Determination of In Vivo Phagocytosis (Lambda Assay)

One-mL aliquots from each of the individual or paired lavages were analyzed for radioactive gold ( 1 9 8 A ~ ) content. The lungs and trachea were removed following BAL to determine the amount of radioactivity remaining, which was reported as gold retention. The radioactive content of each sample was quantitated on an automatic gamma well counter (Packard Instruments, Downers Grove, IL). The total number of cells and radioactivity from all 12 lavages were determined, and the amount found in each sample was expressed as a percentage of this total (100%). Normalization of the data in this way was important so that washout curves of cells and radioactivity could be mathematically analyzed.

To characterize the extent of particle endocytosis, we calculated the parameter lambda (A) using the mathematical analysis described by Brain

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748 C. W. Frevert et al.

and Corkery [20]. Lambda ( A ) is the fraction of the instilled gold which is macrophage associated. Briefly, A is calculated using the equation below which compares the washout pattern of macrophages (macrophage numbers denoted by C) and colloidal gold (19*Au activity denoted by X ) in each of the 7 individual and paired lavage samples (i denotes the lavage samples). Free particle washout (denoted by G) was determined in control experiments where BAL was performed immediately after the instillation of 1 9 8 A ~ in order that almost no phagocytosis of gold would occur. If the I9'Au content in the lavages resembles the cell washout pattern, it is assumed that a high degree of phagocytosis has occurred (A approaches 1 ) ; however, if the washout pattern of 9 8 A ~ resembles the free-particle washout, then little phagocytosis has occurred (A approaches 0).

(Gi - Xi) (Gi - Ci) (Gi - C i ) 2

A=C

Albumin Assay

Albumin levels in the BAL were estimated from lavages 1 and 2. The 2 combined lavages were centrifuged (Beckman GPR, Fullerton, CA) at 4009 and 4°C for 10 minutes. The supernatant was removed and centrifuged (Sorval RC-5, Wilmington, DE) at 11,000 rpm and 4°C for 30 minutes. The cell-free supernatant was stored at - 20°C for later analysis.

The measurement of albumin was performed following the procedures described by Doumas and Biggs [21]. In this assay albumin levels were deter- mined by the binding of bromcresol green. Samples were read at 630 nm on a spectrophotometer (Gilford Response Spectrophotometer, Oberlin, OH) with bovine serum albumin used as a standard.

Statist ica I Ana I ysis

We have focused the statistical analysis on detecting a trend with increas- ing LPS doses. We wanted to know whether LPS increased or decreased AM phagocytic function. T o determine if there was an overall effect of LPS on the parameters measured (i.e., AM phagocytosis, AM and PMN numbers, and albumin levels), a multivariate analysis was done. To study the effects on the individual parameters, a regression line was fit to the data and tests were performed to determine whether the slopes were different from zero. The statistical package SAS (SAS Institute Inc., Cary, NC) was used to fit this model.

For some of the parameters, the linear regression model did not ade- quately fit the data. Therefore, we have also evaluated our results with the nonparametric test for trend developed by Jonckheere and Terpstra [22] to

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Endotoxin Enhances Macrophage Phagoytosis

0.2

0.0 -

749

r

A Slope = 0.0535 (p=0.0062) 1

E y 0.4

0.8 1

0

8 0

0 0

E y 0.4

0

8 0

Slope = 0.0149 (p=0.2839) B

1.0 1

0.8 I 0 0

0 0

I 0 C

0

0 2 4 6 8 10 lntratracheal Dose of LPS (mg/kg)

FIGURE 1 In \;ivo alveolar marrophage phagocytosis of 19'Au following IV ( ,A) and I T (B) LPS admin- istration. [A) With increasing doses of IV LPS. enhanced AM phagocytosis (P = .006) was observed. ( B ) Even at the highest dose of IT LPS, AM phagocytosis was not altered (P = .284). Each circle indicates an individual animal.

analyze our data. This method of statistical analysis is based on the ranks of the data and is less sensitive to unequal variances among groups and to unusually small or large values [22]. Large values of the Jonckheere-Terpstra test statistics are indicative of a dose-response trend and result in rejection of

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750

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C. W. Frevert et al.

A

.- b 0 Slope = -0.05713 (p=0.0165)

0 a, >

0 1 2 3 4 5

intravenous Dose of LPS (mglkg)

a 0 h 2 ! o , , , , ,

B

b v

0 a, il > 2 l a 0

Slope = -0.3310 (p=0.0015)

0

0

0 2 4 6 a 10

intratracheal Dose of LPS (mg/kg)

FIGURE 2 Alveolar macrophage numbers in BAL following IV (A) and IT (B) LPS administration. As IV and IT doses of LPS were increased, there was a decreased recovery of AMs in BAL. Each circle indicates an individual animal.

the null hypothesis of no trend. StatXact software (Cytel Software Corpora- tion, Cambridge, MA) was utilized to compute the exact P values for the Jonckheere-Terpstra test. Results were considered significant when the P values were less than .05.

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Endotnxin Enhances Macrophage Phagncytnsis 75 1

A Slope = 0.0808

0 0

0

0.0 0 1 2 3 4 5

Intravenous Dose of LPS (mg/kg)

Slope = 1.1614 (p=0.0120) 0 0

0 0

O J ? 0 2 4 6 8 10

lntratracheal Dose of LPS (mg/kg)

FIGURE 3 Neutrophil recruitment into alveoli following IV (A) and IT (B) LPS administration. There was an increasing number of PMN in BAL when LPS was administered IT (P = .012), but not IV (P = ,148). Note that the scale of the vertical axis in (A) is different from that in (B) . Intratracheal LPS elicited a far greater PMN response than did intravenous LPS. Each circle indicates an individual animal.

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752 C. W. Frevert et al.

A Slope = -6.5732 (p=0.1400)

0

0 1 2 3 4 5

Intravenous Dose of LPS (mg/kg)

B

1500 I 1250

h - \ E 1000 0) 5 v ,= 750 E = 500 0

.-

a 250

Slope = 38.481 1 (p=0.0552) 0

1 0

0 1 , 0 2 4 6 8 10

lntratracheal Dose of LPS (mg/kg)

FIGURE 4 Albumin levels in the cell-free fraction of BAL following IV (A) or IT (B) administration of LPS. There was no significant increase in albumin at any dose of IV or IT LPS when compared to control, Note that the scale of the vertical axis in (A) is different from that in (B). Each circle indicates an individual animal.

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Endotoxin Enhances Macrophage Phagogtosir 753

The log of the neutrophil and AM cell counts, which resulted in more equivalent variances among the groups, was analyzed. Because there was no difference in the results when the log transformation was used, the discussion below utilizes analysis of the untransformed cell counts.

R ES U LTS

Utilizing multivariate analysis for both the IV and IT treatment groups, we found that LPS had an effect on the 4 parameters measured (i.e., AM phagocytosis, recovery of AMs, PMN, and albumin with BAL) ( P < .05). T o study the effect of LPS on individual parameters, a parametric (i.e., linear regression) and a nonparametric (i.e., Jonckheere-Terpstra test) method of statistical analysis were utilized. Because the conclusions of both statistical analyses were the same, we have used linear regression for our discussion.

T o evaluate the effect of endotoxin on AM phagocytosis, the lambda assay was performed 24 hours after the administration of LPS. We found that the IV administration of LPS caused a significant dose-related increase ( P = .01) in AM phagocytosis of 1 9 8 A ~ (Figure 1A). In contrast, the IT administration of endotoxin failed to have a significant effect on AM phago- cytosis (Figure 1B). When evaluating individual data points in Figure IB, it was noted that in the group administered IT LPS at 5 mg/kg, two animals with low values for lambda depressed the mean of this group. The median, being more robust t o outliers, was not affected by these two low values.

To detcrmine if LPS could influence the composition of alveolar cell populations, both total and differential cell counts were performed on the BAL recovered from rats 24 hours after LPS pretreatment. Across all doses of LPS given 1V ( P = .016) and IT ( P = .001), the number of AMs recovered in the BAL decreased significantly (Figure 2) . In contrast, there were increas- ing numbers of PMN in BAL when LPS was administered IT ( P = .006) (Figure 3).

Albumin levels in the cell-free fraction of BAL were determined and used to assess changes in vascular permeability (Figure 4). There were no signifi- cant increases in albumin levels across all levels of IV (P = .1628) or IT (P = .055) LPS. Note, however, that albumin concentrations were generally higher in the IT LPS group. Considerable variability may have made it less likely to achieve a significant result.

DISCUSSION

We have characterized in situ AM phagocytic function in the rat follow- ing either IV or IT administration of LPS. This was done because we won- dered whether the susceptibility of certain patient populations to bacterial pneumonia might be explained by LPS-induced impairment of AM phago- cytic function. In vivo AM phagocytosis was estimated using a technique

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devised by Brain and Corkery [20] which characterizes the washout patterns of uningested particles and cell-associated particles in BAL. This assay has been used in the past to measure the in vivo effect of a number of substances on AM phagocytosis [23, 241.

We have shown that IV administration of LPS resulted in increased in vivo AM phagocytosis (Figure 1A). Even high doses of endotoxin given I T did not compromise AM phagocytic function (Figure 1B). This is in contrast to the findings of others who have shown decreased AM phagocytic function following IV endotoxin administration [16-181. A significant advantage of the lambda assay is that it measures AM phagocytosis in vivo. Except for the work of Shennib et al. [18], other studies showing decreased AM phago- cytosis have utilized in vitro assays following in vivo exposure [16, 17, 191.

Even though Shennib and colleagues [18] found decreased in vivo AM phagocytosis in their pig model of acute lung injury, they suggested that AM dysfunction was not intrinsic but due to changes in the milieu of the macro- phages. They based this assumption on their findings of decreased in vivo AM phagocytosis but normal in vitro AM phagocytosis. Because we found no significant changes in BAL albumin levels following either the IV or IT administration of endotoxin (Figure 4), we have no evidence that endotoxin caused pulmonary edema, although mediator concentrations may have changed. Pigs are more sensitive to endotoxin and edema is a more likely outcome. Thus although our in vivo findings differ from those of Shennib and co-workers [ 181, our findings are consistent with the conclusions of Shennib et al.

The fact that our study utilized the rat as an animal model and Shennib et al. [18] studied the pig may be significant. There are important differences between the mononuclear phagocyte systems (MPS) of these two species [25, 261. Specifically, the pig has abundant pulmonary intravascular macro- phages (PIMs) whereas the rat lacks these cells [27, 281. Species differences in the MPS have been shown to influence the organ localization of IV LPS and the subsequent inflammatory response [29]. A consistent observation made with direct comparisons between species that have PIMs (ix., pigs, sheep, cat) versus those species that lack these cells (i.e., rat and dog) is that the former show increased susceptibility to endotoxin-induced lung injury [29-321. Thus, in the work of Shennib et al. [18], we hypothesize that in the pig, PIMs may have contributed to the development of endotoxin-induced lung injury and to profound alterations in the alveolar milieu. In contrast, for our studies in the rat, changes in the alveolar milieu are less likely.

The phagocytosis of particles by the AM in vivo is considered to be a 3-step process with the first step being contact, the second step being receptor binding, and the third step being internalization of particles. Of these 3 steps, contact between the AM and the gold colloid and the requirement for motil- ity are probably rate-limiting in phagocytosis, based on a mathematical

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Endotoxin Enhances Macrophage Phagoqtosis 755

model developed for macrophage/bacterium encounter in two dimensions [33]. Although the lambda assay is considered a phagocytic assay, it in fact measures AM mobility, because speeding up or slowing down macrophage movement would directly affect the progression of phagocytosis by either increasing or decreasing the probability of AM particle contact respectively. Therefore, one potential explanation for the increased AM phagocytosis in endotoxemic rats could be increased AM mobility.

Another explanation for the increased AM phagocytosis of gold colloid is LPS-induced changes in receptor numbers on the cell surface. Increased numbers of receptors could increase the number of bound gold particles (i.e., the second step of phagocytosis) and ultimately AM phagocytosis. Unfor- tunately, the receptors responsible for phagocytosis of unopsonized particles have not been well characterized. One potential receptor for AM phago- cytosis of gold colloid is the scavenger receptor which has been shown to bind to gram-positive bacteria [34] and to environmental particulates [35]. Increased expression of scavenger receptors on AM in endotoxemic rats is highly unlikely because both LPS and tumor necrosis factor (TNF)-a have been shown to selectively inhibit the expression of these receptors on human monocyte macrophages [36, 37).

Based on our data, we make the following conclusions. First, endotoxin does not directly inhibit in vivo AM phagocytosis. Second, the increased AM phagocytosis observed in endotoxemic rats is most likely caused by increased AM mobility. Finally, the alveolar milieu can be an important determinant of AM function in vivo.

Following either the IV or I T instillation of LPS, there were decreases in the number of AMs (Figure 2 ) recovered in the BAL. The decreased recovery of AMs could result from either an increase in macrophage adherence (less efficient recovery) or from macrophage loss due to either apoptosis or nec- rosis. Although LPS has been shown to cause AM apoptosis in vitro [38], we feel that the most likely cause of the decreased recovery of AMs 24 hours following LPS is increased adherence of AMs. This conclusion is based on the following 3 reasons. First, LPS causes an increased adherence of rat AM to an alveolar type I1 cell line in vitro [39]. Second, the decreased recovery of AMs in BAL of endotoxemic rats has been shown to be the result of an increase in AM adherence in vivo [40]. Finally, if endotoxin had caused macrophage apoptosis or necrosis, we believe that AM phagocytosis would have decreased, not increased. We therefore conclude that the IV or IT administration of LPS causes increased adherence of AMs, resulting in a decrease in their recovery with increasing dose of LPS.

When BAL fluid albumin levels were measured, there was no significant increase in albumin levels across all levels of IV and I T LPS (Figure 4). Although these findings are unexpected, they were not unprecedented. Three separate studies in human [41], rats [42], and sheep [43] have shown that

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BAL protein concentrations correlated only weakly with BAL PMN numbers. Based on these data, we conclude that the lack of albumin in the BAL fluid suggests LPS is able to recruit PMN into the air spaces of the lungs without causing significant lung injury or long-term changes to epithelial cell permeability.

CONCLUSION

We have shown that the IV or IT administration of endotoxin does not diminish AM phagocytosis in the rat. Although LPS does not have a direct role in decreasing AM phagocytic function, there is evidence that endotoxin can indirectly decrease AM phagocytosis by altering the alveolar milieu in other species. Thus the nature and severity of LPS-induced injury and inflammation in the lungs are critical in determining the extent to which LPS alters host defenses. Further study needs to be done on the ability of endo- toxin to adversely affect pulmonary host defenses.

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