JOURNAL OF IMMUNOlOGICAL METHODS

Journal of Immunological Method\ 193 ( 1996) 149- I56

Assessment of in vivo attachment/phagocytosis by alveolar macrophages

Todd Weaver, Chris L. Hall, Diane L. Kachel, Robert P. Ward, Mark D. Williams, Douglas G. Perry, Paul Wisniowski, William J. Martin II *

Received I8 July 1995: revised 30 October 1995: accepted 5 February I996

Abstract

Alveolar macrophages CAMS) are recognized as an important first line of cellular host defense within the lung. Although mechanisms underlying AM response to microorganisms or particulates are well characterized in vitro. experimental approaches to the study of AMs in vivo are limited. To circumvent these limitations, a new assay was developed using fluorescently labelled liposomes or P~rumoc~sfis cctrinii (PC) organisms which were administered intratracheally into mechanically ventilated rats. After 30 min. the lungs were lavaged and the percentage of administered liposomes or PC bound to AMs was determined by quantifying fluorescence. Factors known to enhance attachment/phagocytosis by AMs in vitro were assayed to determine their effect in vivo. For example, vitronectin (VN)-coated liposomes increased attachment from 25.2 & 2.4’3 to 47.2 f 3.0% (p < O.OOl), while addition of VN increased the binding of PC to AMs from 16.5 f 1.7% to 24.5 li_ 2.27r f p < 0.05). Confocal laser microscopy of cells obtained by lavage provided morphologic evidence of attachment/phagocytosis by AMs. This model will permit the quantitative assessment of the interaction of fluorescently labelled liposomes or microorganisms with AMs in the lower respiratory tract of living animals.

Kryvords: Liposome: Pmwmocystis mritlii: Vitronectin; In viva: Phagocytosis: Adherence

1. Introduction

The respiratory system has a variety of mecha- nisms to facilitate clearance of inhaled particulates or

Abbreviations: AMs. alveolar macrophages: EDTA. ethylene-

diamine-tetradcetic acid: FITC. fluorescein isothiocyanate: HBSS,

Hanks’ balanced salt solution: Hepea, N-Q-hydroxyethyl)pipera-

zinc-N’-(ethanesulfonic acid): PBS, phosphate buffered saline:

PC, Pnertrnocystis carinii: VN. vitronectin. _ Corresponding author. At: Division of Pulmonary and Critical

Cure Medicine. Indiana University School of Medicine. 1001 West Tenth Street. OPW 325. Indianapolis. IN 46202-2879. USA.

Tel.: (317)630-E-145; Fax: (317)630-6386.

microorganisms which enter the lower respiratory tract. Particulates or microorganisms which reach the distal airspaces of the lung initially come into con- tact with alveolar macrophages CAMS) which repre- sent the first line of cellular defense within the lung. Upon association with inhaled particulates. AMs have the ability to initiate opsonin-independent phagocy- tosis (Parod and Brain, 1986: Kobzik et al., 1990). Additional AMs are recruited to the site of infection by release of chemotactic factors (Fisher et al., 1988; Warheit et al., 1988) which amplify this initial host response. Following phagocytosis, the particulates or microorganisms ingested by the AMs are then cleared

0023-1759/96/$l5.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved

P/I soo21- I759(96)0003 l-2

from the lung, primarily through the conducting airways (Lehnert, 1992).

Phagocytosis by AMs has been studied using a variety of methods. Mechanisms underlying phago- cytic cell attachment and phagocytosis have largely been determined using in vitro assays (Absolom. 1986; Wright, 1986) although there is some concern regarding altered AM function in vitro (Fels and Cohn, 1986). AM phagocytosis and clearance has been studied in vivo using a variety of insoluble particles, including ferromagnetic particles (Gehr et al., 1983) and iron oxide (Sorokin and Brain, 1974; Brain et al., 1984). While much has been learned from these studies. there are limitations. These meth- ods are not easily adaptable to the study of microor- ganisms, may require specialized equipment not pre- sent in conventional laboratories. and the interaction with AMs may not be easily quantifiable.

Thus, we have developed a novel in vivo method to measure AM interaction with either liposome-en- capsulated fluorescent latex microspheres or fluores- cein isothiocyanate (FITCH-labelled Ptzeutnoc~stis caritzii (PC) organisms. Liposomes were chosen for a variety of reasons including: (I) ease of quantify- ing the number of liposomes attaching to AMs. (3) the ability of liposomes to incorporate proteins within their phospholipid bilayer, permitting the study of AM attachment and phagocytosis modulation, and (3) opportunity to alter surface charge on liposomes which permits the study of particle charge on phago- cytic mechanisms (Gonzalez-Rothi et al.. 1991; Pa- tel. 1992). FITC-labelled PC organisms were used as a model of airway-borne infectious organisms which are the initial responsibility of AMs to recognize and phagocytose (Fels and Cohn. 1986; Lehnert. 1992). This study demonstrates the feasibility and potential to examine attachment/phagocytosis by AMs in liv- ing animals.

2. Materials and methods

All animal procedures were performed in accor- dance with the Guide for Care and Use of Labora- tory Animals and were approved by the Indiana University School of Medicine Animal Care and Use Committee.

Negatively charged liposomes were freshly pre- pared from egg lecithin, dicetyl phosphate and cholesterol in a molar ratio of 9 : 2: 1 respectively (Avanti Polar Lipids, Alabaster, AL). Briefly, the contents of the liposome kit were dissolved under nitrogen and dried under vacuum. Swelling solution ( 150 mM NaCl, 20 mM Hepes) containing 0.05 pm fluorescent microspheres (Polysciences. Warrington. PA) with or without vitronectin, or with or without PC-gpl?O was added to the dried extracts and then vortexed and freeze-thawed five times to entrap the microspheres and proteins. The suspension was washed five times with swelling solution with se- quential centrifugation (9500 X g, 5 mini in a Sor- vail RC28S centrifuge (DuPont, Hoffman Estates. IL). All liposome preparations were used immedi- ately or stored at 4°C for a maximum of 38 h. Liposome preparations stored in this manner consis- tently retained their label with minimal evidence of clumping.

Vitronectin (VN) used in the preparation of lipo- somes was isolated on a heparin affinity column as previously described (Yatohgo et al., 1988; Mimuro and Loskutoff. 1989). A fibronectin affinity column was used to isolate PC-gpl20 as described by Wis- niowski et al. (1994).

2.2. Isolatiott ad preparatiotl of PC

PC infection was induced in female 150-200 g Harlan Sprague Dawley rats (Harlan Sprague Daw- ley. Indianapolis, IN). The rats were housed in open cages, supplied with rat chow (Teklad 7001) and drinking water supplemented with dexamethasone (2 mg/lI ad libitum. After 2 weeks, the rats were inoculated with 5 X lOh PC organisms intratra- cheally to enhance infection (Boylan and Current. 1992). After 6 weeks. the rats were killed with pentobarbital sodium (200 mg per rat) (Schering- Plough Animal Health, Keniworth, NJ).

Bronchoalveolar lavage was performed using Hanks’ balanced salt solution (HBSS) without Ca’+ supplemented with 0.6 mM EDTA. penicillin (100 U/ml). streptomycin ( 100 ~g/mlI. amphotericin (0.025 pg/ml) and gentamicin (4 pg/ml). Instilla- tion of five sequential aliquots of 10 ml each were performed for a final return of approximately 45 ml.

T. Wmwr et cd. / Joun~al of Immunological Methods I93 (1996) 149-156 151

PC were isolated from the lavage fluid by differ- ential centrifugation as previously described (Pottratz and Martin, 1990). Briefly. lavages were centrifuged (400 X g, 10 min) to pellet inflammatory cells. Cyto- preparation smears were prepared in a Shandon Cy- tospin 2 (Shandon Southern Instruments, Pittsburgh, PA). Cytopreparation slides were stained with Diff- Quik (Biochemical Sciences, Bridgeport, NJ) and viewed under oil immersion. If there was any evi- dence of microorganisms other than PC in a lavage specimen, the specimen was immediately discarded. Lavage supematants were centrifuged ( 1400 X g, 30 min) to pellet PC trophozoites. The pellets were pooled and resuspended in 5 ml HBSS. PC organ- isms were quantified by the method of Bartlett (Bartlett et al., 1979). To further ensure the absence of contaminating bacteria in the PC isolates from the retained lavage samples, aliquots of the PC samples were cultured in blood heart infusion broth (Difco, Detroit, MI) for an overnight incubation. If there was any evidence of growth, the PC isolate was not used for experimentation.

PC were labelled with FITC according to the method of Ezekowitz et al. (1991). In brief, PC were incubated in 0.1 mg/ml FITC in phosphate buffered saline (PBS) (pH 7.4) at a concentration of 10’ PC/ml for 30 min at 37°C. The PC suspension was centrifuged (1400 X g, 30 min) followed by two subsequent 20 min washes with PBS at room tem- perature to sufficiently remove any free or unincor- porated FITC label. PC were inoculated directly or after pre-incubation with VN (5.1 PM) in PBS in the presence of Ca’+ (5.0 mM). After 30 min the PC were washed (1400 X g, 30 min). Selected samples were incubated with anti-VN antibody F(ab’)? frag- ments (0.055 mg/ml) for 10 min. Anti-VN F(ab’)? fragments were prepared by standard methods (Pierce Chemical Co., Rockford, IL).

2.3. In rice model of AM attachment/phagocytosis

Harlan Sprague Dawley rats (210-250 g) were sedated with 30 mg ketamine HCl/kg body weight. After the animal had reached a deep level of seda- tion, the trachea was exposed using blunt dissection. The rat was orally intubated with a 14 gauge angio- cath (Becton Dickinson. Sandy, UT) which was se- cured with 4-O silk. The animal was paralyzed with pancuronium bromide (0.1 mg/kg body weight) and

ventilated at the recommended minute ventilation of 100 ml/min (Bivin et al., 1979) using a small animal respirator (Analytical Specialties Co., St. Louis, MO). This ventilator model permitted ventila- tion of two animals simultaneously. The peak inspi-

ratory pressure was set at a maximum of 16 cm H,O with a positive end expiratory pressure of 1 cm H,O with frequency set at 56 breaths/minute with an I: E ratio of 1 : 2. This resulted in a tidal volume of approximately 1.8 ml. Following a 30 min acclima- tion to the ventilator, rats were instilled with 8.0 X 10’ fluorescent liposomes or 2.0 X 10’ FITC-labelled PC in a volume of 200 ~1 PBS intratracheally via a 3-way stopcock with the rats in a supine position. Instillation of liposomes or PC was timed during inspiration to aid in distribution of the agents to the lower respiratory tract. After 10, 30, 60, or 120 min, the animal was killed by exsanguination and lavage of the lungs was immediately performed with five aliquots of 8 ml HBSS without Ca’+ containing 0.6 mM EDTA. Lavage returns were typically 37 ml of the 40 ml instilled. The total number of liposomes or PC obtained by lavage was determined by measure- ment of fluorescence in duplicate 250 ~1 aliquots using a CytoFluor fluorescent plate reader (Milli- pore, Bedford, MA). The fluorescence was compared to a standard curve of fluorescent liposomes or FITC-labelled PC to determine the quantity of lipo- somes or PC in the sample. The remaining lavage fluid was centrifuged to pellet AMs and their associ- ated liposomes or PC (400 X g. 10 min). Supematant was removed until 5 ml remained with the pellet. The pellet was resuspended in the 5 ml and cyto- preparation smears were made and stained with Diff-Quik stain. All samples contained > 98% AMs in the cell differential. The number of AMs was determined using a hemocytometer. Duplicate 250

~1 aliquots were removed from the supematant and pellet fractions and measured with the CytoFluor plate reader. Supematant readings had to be sub- tracted from pellet readings to compensate for fluo- rescence in the remaining supematant over the pellet. Liposome or PC number in the samples were deter- mined as described above. Data is presented as: (1) percent liposomes or PC associated with AMs = number of liposomes or PC in pellet/(number of liposomes or PC in pellet + number of liposomes or PC in supematant) and (2) percent return = the num-

ber of liposomes or PC in the whole return/total amount of liposomes or PC instilled.

After ventilation. selected rat lungs were perfused with sterile PBS via the pulmonary artery. These lungs were sectioned into multiple pieces from cephalad to caudal regions. The pieces were weighed and homogenized. The samples were centrifuged (400 X g, 10 min) to pellet tissue debris. Fluores- cence from the supematant (250 ~1 aliquots) was determined using the CytoFluor. The evenness index (Brain et al.. 1976) for each piece was determined as [(fluorescence/g wet weight,,,,,)/(fluorescence/ gram wet weight who,e )] X 100. If the fluorescence was evenly distributed, each piece would receive an evenness index of 100. An evenness index < 100 means the lung piece received less than its expected fluorescence. Likewise, an evenness index > 100 means the piece received greater than its expected fluorescence.

Similarly, selected rat lung and rat lavage speci- mens were viewed by confocal laser scanning mi- croscopy (Inout, 1990; Pinkerton et al., 1993: Wright et al., 1993). Confocal microscopy permitted mor- phologic detection of fluorescent liposomes or PC

organisms from lavage (cytopreparation smears) or in situ in the lung (fresh, unfixed rat lung specimens).

2.4. Statistical analwis

Differences in % return of liposomes were ana- lyzed by ANOVA followed by Newman-Keuls pair- wise comparison, and % liposome or FITC-PC-asso- ciated AMs were analyzed by Student’s t test. Vari- ance between o/c attachment/phagocytosis after in- stillation of liposomes or PC in different animals was analyzed by determining the coefficient of variance. All data represent the mean + SEM.

3. Results

3. I. Distribution of liposomes and PC in the lung

The distribution of liposomes and PC in the lung following intratracheal administration into mechani- cally ventilated rats in the supine position was ex- pressed as an ‘evenness index’. This reflected the % of fluorescent liposomes (Fig. la) or FITC-PC (Fig. I b) in each section of the lung as a function of

30 a.

,.5111 1 2 3 4 5 6

cl”

-j 20

b.

1 ,oI; 1 2 3 4 5 6 7

Fig. I. Distribution of fluorescent liposomes. Ventilated wts were

intratracheally inoculated with (LI) 8.0X IO’ tluorexzent liposomeb

in 200 ~1 of PBS or (6) 3.0X IO’ FITC-PC in 200 ~1 PBS. After

30 min the rats were killed and the lung was divided into seven

hectionh from cephalad to caudal regiona. The wet weight of the

specimen wa> determined. the fluorexence was quantified and the

evennes index was calculated as described in Section 2. The data

indicated that intratracheal administration resulted in a higher

distribution of the liposomes in the more caudal \ectionh of the

lung.

cephalad-caudal distribution. If the liposomes or PC were evenly distributed, the evenness index of each section would have a score of 100 (Brain et al.. 1976). In both cases, the data indicated that the evenness index progressively increased from lowest concentration of liposomes or PC in the cephalad sections of the lung to the highest concentration in the most caudal lung sections which had an index of 218.6 k 3 1.7 or 147.8 + 16.6, respectively.

3.2. In c,iLw clttuchmerlt/ phagoc:vtosis of lip~~sonws

by AMs

The %return of fluorescent liposomes was re- markably reproducible. Initial studies using sedated,

153

0 30 60 90 120 150

Time (min.)

Fig. 2. Effect of time on tic of liposomes obtained by lavage

compared to total number instilled. 8.0X IO’ fluorescent lipo-

somes were instilled into the airway of mechanically ventilated

rats. The percent of liposomes returned in a lavage remained

consistent at 30 minutes and beyond as determined by ANOVA

followed by Newman-Keuls pairwise comparison ( p < 0.05).

but non-mechanically ventilated rats, resulted in highly variable %return (data not shown). The per- centage of returned liposomes decreased from 40.6 _t 1.0 at 10 min to 32.2 + 1.9 at 30 min (Fig. 2). At 30, 60, and 120 min; however, there was no signifi- cant difference in the % return of liposomes (p > 0.05). The 30 min time point was chosen for subse- quent studies as the return was reproducible from animal to animal, significant attachment/phagocyto- sis by AMs occurred, and the % return was un- changed from 30 to 120 min indicating no complicat- ing factors in vivo were yet occurring (loss of fluo- rescence, clearance of the liposomes from the lower respiratory tract, etc.).

Attachment of fluorescent liposomes (without in- corporated proteins) to AMs was 25.2 + 2.4 (Fig. 3). Recent studies suggest adhesive proteins such as VN may act as a nonimmune opsonin in macrophage phagocytosis (Savill et al., 1990; Fadok et al., 1992). When VN was incorporated into the phospholipid bilayer of the fluorescent liposomes. liposome at- tachment/phagocytosis by AMs in vivo increased from 25.2 + 2.4% to 47.2 + 3.0% ( p < 0.001 I. However, it is possible that protein-incorporated li- posomes may have a higher % return than nonpro- tein liposomes and, thus, have an effect on the % attachment. In our experiments with VN-incorpo- rated liposomes, there was a slight increase in the % return of the VN-incorporated liposomes compared to the control liposomes; however, this difference was not significant ( p = 0.08) (data not shown).

Blank VN GP 120

Type of Liposomes

Fig. 3. Effect of VN or PC-gpl20 on percent of lipoaomes

associated with AMa. Lipoaome attachment to AMs in viva

increased with addition of VN or PC-gpl20 to the phospholipid

bilayer. Results are expressed as mean + SEM (Srp < 0.05; tltp

< 0.001).

Since in vivo attachment/phagocytosis of VN liposomes by AMs was increased. this suggested the feasibility of studying the role of nonimmune op- sonins and AM defense mechanisms in vivo. Simi- larly, liposomes could be made which contain known glycoproteins from the surface of microorganisms to determine if AMs recognize these cell surface mark- ers. In this regard, PC-gp120, the major cell surface glycoprotein on PC. was incorporated into the lipo- somes and the attachment/phagocytosis was in- creased from 25.2 k 2.4% to 34.4 + 1.9 at 30 min

( p < 0.05). As described for fluorescent liposomes, the at-

tachment/phagocytosis of FITC-labelled PC organ- isms by AMs in vivo could be easily quantified (Fig. 4). 30 min following intratracheal administration of FITC-PC, the percent of PC-associated AMs re- turned in bronchoalveolar lavage was 16.5 + 1.7%. VN pre-incubation with the PC increased the percent of PC-associated AMs from 16.5 * 1.7% to 24.5 k 2.2% (p < 0.05). Incubation of an F(ab’I, fragment of a monoclonal anti-VN antibody blocked this en- hanced attachment of PC from 24.5 k 2.2% to 13.6 & 4.7%. This indicated the interaction of fluores- cently-labelled microorganisms with AMs could be modulated in vivo; thus, this model system offered unique opportunities to test hypotheses developed by in vitro studies in the living animal.

Over a period of 5-6 different days, studies were conducted to assess the variance in the in vivo

Fig. 1. Effect of VN on AM-associated PC. PC attachment to

AMs in viva was increased after pre-incubation with VN and was

decreased to control levels in the presence of anti-VN antibody

F(ab’), fragments. Results are expressed as mean+SEM (+p <

0.05).

% attachment/phagocytosis of liposomes or PC by AMs on an animal to animal basis. 30 min after instillation, liposomes and PC resulted in a % attach- ment/phagocytosis by AMs of 25.2 f 2.67~ and 19.2 + 1.7%. respectively, with both sets of data having a coefficient of variation of approximately 27%.

3.3. C0nfocal microscopy to assess attachment/ phagocytosis by AMs in l’irw

To provide morphologic evidence of AM attach- ment/phagocytosis of the FITC-PC in vivo, selected lavage and lung specimens were viewed by confocal microscopy (Fig. 5). Clearly, the red auto-fluores- cence of AMs in both specimens identified this cell type and permitted morphologic detection of attach- ment/phagocytosis of the brightly yellow-green flu- orescent FITC-PC organisms. Confocal microscopy permitted cross-section of the AMs at incremental intervals from 0.5 to 1 Km, thereby distinguishing

Fig. 5. A: confocal microscopy of PC distribution in lung alveoli. 2.0X IO’ FITC-PC were instilled into mechanically ventilated

mts. After 30 min the animal was killed and the lung removed and viewed using confocal microscopy. Yellow-green FITC-PC are

readily visualized within the alveolar spaces (note green autofluo-

rescence of alveolar walls) with and without coming into contact

with red autotluorescent AMs (600x ). B: cytopreparation smear

of a rilt lavage clearly shows yellow-green FITC-PC attached and ingested by red autofluorescent AMs (600 x ).

T. Wearer et 01. / Joumnl of Inzmunological Methods 193 (19961 149-156 155

FITC-PC organisms on the surface of the AM from those inside the AM. Clearly, the data provided morphologic confirmation that significant att.ach- ment/phagocytosis by AMs occurred in vivo as early as 30 min following intratracheal administra- tion in rats.

4. Discussion

For an in vivo model to be useful in the study of attachment and phagocytosis by AMs, the design should be adaptable, quantifiable, physiologically relevant and reproducible. Mechanical ventilation is the key to providing the above components. Ventila- tion of animals has been useful in the development of different model systems examining the lung in both health and disease (Thrall et al., 1987; Connelly et al., 1992). Control of the airway and mechanical ventilation to ensure adequate inspiratory volume are keys to the reproducibility of our model examining AM attachment/phagocytosis in vivo. In other mod- els, variable deposition of particles occurs in the lung, as a significant percentage are cleared by upper airway mechanisms (Ferin et al., 1992). Since rats are obligate nasal breathers, direct instillation of liposomes or microorganisms into the trachea avoids capture of aerosol in the nasal hairs of rodents. Other factors, such as handling and restraining animals. also affect the respiratory pattern of the animal and alter distribution of particles in the lung (Sweeney and Brain, 1991). Most importantly, however, me- chanical ventilation ensures that lung volumes and minute ventilation are maintained in the normal physiologic range (Bivin et al., 1979). In contrast, sedated spontaneously breathing rats exhibit different levels of activity with variation in lung volumes and minute ventilation which result in variable deposition of the instilled material. A consistent level of seda- tion and mechanical ventilation eliminates these problems and ensures a high degree of reproducibil- ity.

Although the advantages of this method are clear, the use of sedation and mechanical ventilation makes this approach more labor-intensive. The ventilation in this study permits only two animals to be venti- lated simultaneously. However, with practice, an in- dividual can use two ventilators at the same time,

permitting four animals to be studied under nearly identical circumstances. If the time following intra- tracheal administration of the fluorescent label is

short, i.e. 30 min, as many as 12 animals can be tested during a 4 h period.

Liposomes represent interesting vehicles by which

to assess attachment/phagocytosis by AMs. The in- corporation of PC-gpl20. the major surface glyco- protein on PC organisms (Pottratz et al., 1991). into

liposomes creates an ‘artificial’ PC organism. The addition of gp120 to liposomes produces a marked increase in the % of liposomes associated with AMs.

As a result, this model permits study of surface glycoproteins from microorganisms which are hy- pothesized to mediate attachment to host cells (Pot- tratz et al., 1991, 19931.

VN is a possible nonimmune opsonin. The VN receptor is prominent on macrophages and has been shown to be involved in phagocytosis (Fadok et al., 1992, Savill et al.. 1990). VN incorporated into liposomes increases the attachment/phagocytosis by AMs compared to liposomes without VN. A recent study indicates VN can bind PC and act as a ligand to promote attachment of PC (Limper et al., 1993). Our study verifies that VN can augment PC attach- ment/phagocytosis by AMs in vivo. More impor- tantly. anti-VN F(ab’), fragment reduces attachment

to control levels. F(ab’), fragments are utilized to avoid the FC-mediated binding of the antibody to AMs, suggesting this approach might have potential therapeutic applicability in vivo.

In summary. we have developed an assay which permits quantification of liposome or PC attach- ment/phagocytosis by AMs in vivo. It offers promise as a method to test hypotheses developed by in vitro studies in the lungs of living animals. Furthermore. it provides a quantifiable method to test the applicabil- ity of possible therapeutic agents which are thought to modulate attachment/phagocytosis of microor- ganisms.

Acknowledgements

The authors would like to thank Dr. Rajamouli Pasula for the anti-VN antibodies.

This study was supported by NIH grants HL43524. HL46647 and HL5 1962.

References

Absolom. D.R. ( 1986) Basic method> for the study of phagocyto-

sib. Methods Enzymol. 1.72. 95.

Bartlett. M.S.. Vcrbanac. P.A. and Smith. J.W. (1979) Cultivation

of Ptwruunc~~.\tis c,trrir~ii with WI-38 cell>, J. Clin. Microhiol.

IO. 796.

Bivin, W.S.. Crawford. M.P. and Brewer. N.R. (1979) In: H.J.

Baker. J.R. Lindsey and S.H. W&broth (Eds.) The Laboratory

Rat. Vol. I. Biology and Diseases. Harcourt Brace Jo-

vanovich. San Diego. CA. p. 73.

Boylan. C.J. and Current W.L. (1992) Improved rat model of

P,lrlrrrlo~!,.sti.s ctrrir~ii pneumonia: induced laboratory infec-

tions in P~~r~tr~loc~~stis-free animals. Infect. Immun. 60. 1589.

Brain, J.D., Knudson. D.E.. Sorokin, S.P. and Davis. M.A. (1976)

Pulmonary distribution of particlch given by intmtracheal n-

btillation or by aerosol inhalation. Environ. Res. I I, 13.

Brain. J.D.. Bloom. S.B.. Valberg, P.A. and Gchr. P. (1984)

Correlation hctwccn the behavior of magnetic iron oxide

particle\ in the lungs of rabbits and phagocyto\is. Exp. Lung

Res. 6, I 15.

Connelly. CA.. Otto-Smith. M.R. and Feldman. J.L. (1991)

Blockade of NMDA receptor-channel by MK-801 alters

breathing in adult rats. Brain Res. S96. 99.

Erekowitz. R.A.B.. Williams. D.J.. Koriel. H.. Armstrong,

M.Y.K.. Warner. A.. Richards, F.F. and Rose. R.M. (1991)

Uptake of Pne~rr~rocv.sti.s ccrrinii mediated by the macrophage

mannose receptor. Nature iS I. 155.

Fadok. V.A.. Savill. J.S.. Haslett. C.. Bratton. D.L.. Doherty,

D.E.. Campbell. P.A. and Henion. P.M. (1902) Different

populations of macrophases use either the vitroncctin receptor

or the phosphatidylserine receptor to recognize and remove

apoptotic cells. J. Immunol. 119. 1019.

Fels. A.O.S. and Cohn. Z.A. (1986) The alveolar macrophage. J.

Appl. Phyhiol. 60. 353.

Ferin. J.. Obcrdorhter. G. and Penney. D.P. (1992) Pulmonary

retention of ultrafine and fine particles in rats. Am. J. Respir.

Cell Mol. Biol. 6. 535.

Fisher. E.S.. Lauffenburger. D.A. and Daniele. R.P. (1988) The

effect of alveolar macrophage chemotaxis on bacterial clear-

ance from the lung surface. Am. Rev. Respir. Dis. 137. I 119.

Gehr. P.. BI-ain J.D.. Nemoto. 1. and Bloom S.B. (1983) Behavior

of magnetic particles in hamster lungs: estimates of clearance

and cytoplasmic motility. J. Appl. Physiol. 55. 1196.

Gonzalez-Rothi. R.J.. Strauh. L.. Cacace. J.L. and Schreier. H.

( I99 I ) Liposome\ and pulmonary alveolar macrophages: func-

tional and morphologic interactions. Exp. Lung Res. 17. 6X7.

Ino@. S. (1990) In: J.B. Pauley (Ed.). Handbook of Biological

Confocol Microscopy. Plenum PI-eyr. New York. p. I. Kohrik. L.. Godle\ki. J.J. and Brain. J.D. (1990) Selective down-

regulation of alveolar macrophage oxidativc response to op-

sonin-independent phagocytosis. J. Immunol. 143. 3311.

Lehnert. B.E. (1092) Pulmonary and thoracic macrophage subpop-

ulations and clearance of particles from the lung. Environ.

Health Perspect. 97. 17.

Limper. A.H.. Standing. J.E.. Hoffman, O.A.. Castro. M. and

Necse L.W. ( 1993) Vitronectin hinds to Pwwmc~stis ccr,-iuii

and mediates organism attachment to cultured lung epithelial

cells. Infect. Immun. 61, 4302.

Mimuro. J. and Loshutoff. D.J. (1989) Purification of a protein

from bovine plasma that binds to type I plasminogen activator

inhibitor and prevents its interaction with extracellular matrix.

J. Biol. Chem. 261. 936.

Parod. R.J. and Brain, J.D. ( 1986) Immune opsonin-independent

phagocyto4\ hy pulmonary macrophages. J. Immunol. 136.

2011.

Patel. H.M. (1993) Influence of lipid composition on op-

\onophagocytosi\ of liposomes. Res. Immunol. 113. 743.

Pinkerton. K.E.. Gallen. J.T.. Mercer. R.R.. Wang. V.C.. Plopper.

C.G. 2nd Tarhington. B.K. (1993) Aerosolized fluorescent

microhpherea detected in the lung usins confocal scanning

laher microscopy. Microsc. Res. Tech. 26. 137.

Pottratz. S.T. and Martin II. W.J. (1990) Mechanism of attach-

ment to cultured mt alveolar macrophqes. J. Clin. Inve\t. 86.

167X.

Pottratl. S.T.. Paulsrud. J., Smith J.S. and Martin II, W.J. (1991)

Pw~rmr~~~~sti.~ arrirzii attachment to cultured lung cells by

Pwumr~c~y.\ti.\ gpl20. a fihronectin binding protan. J. Clin.

Incest. 8X. 403.

Pottratr. S.T.. Paul\rud J.R., Smith J.S. and Martin II. W.J. (1993)

Evidence for ftwurrwc~~sti.s urirrii binding to a cell-free sub-

state: role of the adhesive protein fihroncctin. J. Lab. Clin.

Med. 123. 173.

Savill. J.. Dransfield. I.. Hogg. N. and Halett. C. (1990) Vit-

ronectin receptor-mediated phqocytohis of cell\ undergoing

apoptosis. Nature 333. 170.

Sorokin. S.P. and Brain. J.D. (197-l) Pathways of clearance in

mouse lungs exposed to iron oxide aerosol\. Anat. Rec. IX I.

5X1.

Sweeney. T.D. and Brain. J.D. (1991) Pulmonary Deposition:

determinants and measurement techniques. Toxicol. Pathol.

19, 3x4.

Thrall. R.S.. Swendsen. C.L.. Shannon. T.H., Kennedy. C.A..

Frederick. D.S.. Grunxe. M.F. and Sula\ik. S.B. ( 1987) Corre-

lation of change\ in pulmonary surFa%mt phospholipidh with

compliance in hleomycin-induced pulmonary fibro\is in the

rat. Am. Rev. Respir. Dis. 136. I 1.3.

Warheit. D.B.. Overhy. L.H.. George. G. and Brody. A.R. (1988)

Pulmonary macrophages are attracted to inhaled particlea

through complement activation. Exp. Lung Rea. l-1. 51.

Wright. S.D. ( 1986) Methods for the study of receptor-mediated

phagocytoais. Methods Enzymol. 132. 201.

Wright. S.J.. Centonre, V.E., Stricher. S.A.. DcVrics. P.J.. Pad-

dock, S.W. and Schatten. G. (IYY3) Introduction to confocul

microscopy and three-dimensional reconstruction. Methods

Cell Biol. 8. I.

Wisniowaki. P.. Pasula, R. and Martin 11. W.J. ( 1994) Isolation of

f,,c,lr,,io~,~.~t;.\ ctrriuii gp I20 hy fibronectin affinity: evidence

for manganese dependence. Am. J. Respir. Cell Mol. Biol. I I.

761. Yatohro. T.. Izumi. M.. Kashiwagi. H. and Huyashi, M. (1988)

Novel purification of vitronectin from human plasma by hep-

arin affinity chromatography. Cell Struct. Funct. 13, 281.