INFECrION AND IMMUNITY, Mar. 1993, p. 955-9650019-9567/93/030955-11$02.00/0Copyright © 1993, American Society for Microbiology

Ultrastructural Analysis of Attachment to and Penetration ofCapillaries in the Murine Pons, Midbrain, Thalamus, and

Hypothalamus by Nocardia asteroidesBLAINE L. BEAMAN* AND STEVEN A. OGATA

Department of Medical Microbiology and Immunology, University of CaliforniaSchool of Medicine, Davis, California 95616

Received 29 June 1992/Accepted 28 December 1992

The attachment to and penetration of endothelial cells in the pons and midbrain (especially the substantianigra) regions of the brains of BALB/c mice by log-phase Nocardia asteroides GUH-2 cells were determined byboth scanning and transmission electron microscopic analysis. Within 15 min after exposure, the nocardiaeattached to the surface of the endothelial cell membrane. This attachment occurred primarily at the growingtip of the nocardial filament, and the outermost layer of the nocardial cell wall had regions (electron-denseareas) that bound firmly to the cytoplasmic membrane of the host cell. There appeared to be specificity for thisbinding localized within the capillaries and arterioles because some regions had large numbers of bacteriabound, whereas adjacent areas had no bacterial cells. Nocardial filaments that attached by the apex induceda cuplike deformation of the endothelial cell membrane. This was followed by a rapid penetration of theendothelial cell so that within 25 min many of the bacteria were internalized within the host cell. Theseinternalized bacteria remained within vesicles, and there was no ultrastructural evidence of damage to thenocardial cell during this process. Heat-killed GUH-2 cells still attached to endothelial surfaces (at a reducedfrequency), but they did not penetrate into the endothelial cell. These data suggest that brain-invasivenocardiae possess both an adhesin for attachment to the membrane of endothelial cells and an invasion factorthat promotes nocardial penetration of these cells.

Nocardia asteroides is a facultative intracellular pathogenthat causes pulmonary infections in both normal and immu-nocompromised hosts (1). It is well established that thenocardiae can spread by way of the bloodstream to otherregions of the body and that the brain is the major target forinfection during dissemination (1). As a consequence, No-cardia spp. should be considered a brain pathogen in humansand animals (1). Furthermore, murine models for systemicnocardiosis after bloodborne promulgation have been estab-lished (1).N. asteroides GUH-2 was isolated from a fatal human

infection, and its pathogenesis has been studied extensively(2-5). After an intravenous injection of log-phase GUH-2cells into mice, there is a rapid invasion of the brain in 100%of the animals (7). If more than 500 CFU are depositedinitially into the brain after intravenous inoculation, then themice die an acute death due to rapid proliferation of theorganism (7, 11, 12). On the other hand, if fewer than thisnumber of bacteria are introduced into the brain, the animalsmay survive (7). In mice that survive this nonlethal dose, thenocardiae grow silently for several days, and then thenumber of bacteria recovered from the brain decreases.When the brains appear to be sterile, about 10% of the micedevelop a permanent, progressive, neurodegenerative re-sponse that has many features similar to those found inParkinson's disease in humans (7). Light microscopy sug-gests that the bacteria become adherent to capillaries withinspecific regions of the brain. Once attached, these organismsappear to invade the brain parenchyma without causingvisible loss to the integrity of the blood-brain barrier. Fur-thermore, there is no apparent induction of an inflammatory

* Corresponding author.

response at the site of the invasion (7). Viability studiesusing microdissection suggest that log-phase GUH-2 cellspossess a specific adherence mechanism for capillary endo-thelial cells within regions of the brain (11, 12).The purpose of this investigation is to determine whether

there is a specific adherence of nocardiae to the endothelialcell membrane in regions of the brain relevant to the move-ment disorders reported previously (1, 7) and to ascertainhow the nocardiae penetrate through the capillary (11, 12).Therefore, light and electron microscopic analysis was per-formed upon the brains of BALB/c mice that had beenperfused with either live or heat-killed log-phase N. asteroi-des GUH-2 cells.

MATERIALS AND METHODS

Microorganism. N. asteroides GUH-2 was isolated in acase of fatal human infection at Georgetown UniversityHospital, Washington, D.C. (4). This strain has been usedextensively as a model for studying the mechanisms ofnocardial pathogenesis (2-5). It was grown and maintained inbrain heart infusion broth (Difco Laboratories, Detroit,Mich.) as described previously (11).

Inoculum. Suspensions of single cells of N. asteroidesGUH-2 at the log phase of growth were prepared by growthin brain heart infusion broth followed by differential centrif-ugation as described in detail elsewhere (11).As a control on the effect of viability on adherence and

penetration, a suspension of log-phase cells was heated at60°C for 2 h in order to kill the bacteria. The effectiveness ofkilling was determined by direct plating of the suspension on

Trypticase soy agar. The cellular density of the live organ-isms was adjusted to 5 x 106 CFU/ml in minimal essentialmedium supplemented with 1.0% heat-inactivated fetal calf

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serum (GIBCO Laboratories, Grand Island, N.Y.). The deadcells were prepared in the same suspension medium withapproximately the same cellular concentration on the basisof optical density (A580 was measured on a Spectronic 20spectrophotometer).Animals. Female BALB/c mice weighing 18 to 20 g were

obtained from Simonsens (Gilroy, Calif.). The animals weremaintained by the Animal Resource Service at the Univer-sity of California at Davis as previously described (11).

Perfusion of the murine brain. In order to study the earlyevents of attachment and penetration of nocardiae in thecapillaries of the brain, mice were perfused intra-arteriallywith suspensions of nocardial cells. Thus, mice were anes-thetized with 0.5 ml of pentobarbital sodium (Nembutal; 5mg/ml) injected intraperitoneally. The chest cavity wasopened, a needle (27 gauge) was inserted into the leftventricle, and the right atrium was cut. The needle wasattached to Tygon tubing that was placed in a water bath at37°C. The other end of the tubing was inserted into ahomogeneous suspension of log-phase GUH-2 cells (5 x 106CFU/ml) in minimal essential medium supplemented with1.0% heat-inactivated fetal calf serum. The cell suspensionwas perfused through the arterial system for 15 min at a flowrate of 1 ml/min with a peristaltic pump (Buchler Instru-ments, Kansas City, Mo.), and then the arterial system waswashed with 10 ml (1 ml/min) of perfusion saline (9 g of NaClplus 8 g of sucrose plus 4 g of glucose per liter of double-distilled water) in order to remove all unattached bacteriafrom the capillaries. After this wash, 4% (wt/vol) paraform-aldehyde in 0.1 M phosphate buffer (pH 7.3) was perfusedthrough the arterial system for 15 min. Thus, the capillariesin the brain were exposed to nocardial cells for at least 15min but no longer than 25 min before being killed and fixedby the paraformaldehyde. In some experiments, these sameprocedures were repeated except that 2.2% glutaraldehyde(electron microscopy grade; Ted Pella, Inc., Redding, Calif.)was substituted for the 4% paraformaldehyde. Furthermore,some mice were perfused with suspensions of heat-killednocardiae in place of the live suspensions. The perfused,fixed brain was removed, and coronal slices were made. Insome preparations, the pons and substantia nigra regionswere selectively separated from the rest of the brain. All ofthese regions were then prepared for electron microscopy.

Electron microscopy. In some preparations, the previouslyperfused and fixed pons and substantia nigra regions werepostfixed in osmium tetroxide (0.5% in phosphate buffer, pH7.3)-0.5% (wt/vol) uranyl acetate, dehydrated with ethanol,and embedded in Med-cast epoxy resin (Ted Pella, Inc.). Inother instances, the entire midbrain region, including thethalamus and hypothalamus, was postfixed and embedded asdescribed above. After polymerization, thick sections werecut with a glass knife and stained with aqueous carbol-fuchsin (0.2%, wt/vol) to visualize bacteria with the lightmicroscope. When bacterial cells were located, the blockswere trimmed and sectioned for electron microscopy with adiamond knife. Gold-to-silver sections were placed on cop-per Athene grids (200 mesh), stained for 15 min with 0.5%(wt/vol) uranyl acetate in 50% (vol/vol) methanol-water andin 0.1% lead citrate in distilled water (solubilized by addingone drop of 1 N NaOH per 10 ml). The sections were washedin deionized water, dried, visualized, and photographed witha Philips model 400 electron microscope operated at 80 kV.Scanning electron microscopy was used for the visualiza-

tion of bacterial interactions on the endothelial surface inarterioles and capillaries. Thus, after the brain had beenperfused with either live or dead nocardial cells as described

above, the brain was perfused with 2.2% glutaraldehyde.The brain was removed, and coronal slices were made sothat the midbrain and portions of the thalamus and hypothal-amus were exposed. Vibratome sections (100 ,um thick)through these regions of the brain were prepared in aglutaraldehyde bath and placed in fresh 2.2% glutaraldehydeovernight at 4°C. The sections were then washed in phos-phate-buffered saline (pH 7.4), dehydrated through an ofethanol series (starting at 25% [vol/vol] with a gradientincrease to 100% dry ethanol), critical-point-dried with CO2,coated with gold, and examined with a Philips scanningelectron microscope at 15 kV.

Localization of bacteria in the brain. In order to facilitatelocalization of nocardial adherence within the brain, micewere anesthetized and perfused intra-arterially with 5 x 106CFU/ml for 15 min (1 ml/min) as described above. Thenonadherent bacteria were flushed from the brain with 10 ml(1 ml/min) of perfusion saline, which was followed byperfusion with 4% paraformaldehyde for 15 min (1 ml/min) asdescribed above. The brain was removed, coronal sliceswere embedded in paraffin, and 6-pum serial sections wereplaced on glass slides. The sections were stained with eitherhematoxylin and eosin or by the Brown and Brenn modifi-cation of the Gram stain (7). A hematoxylin-eosin-stainedcoronal section that included the substantia nigra, thalamus,and hypothalamus (15) was photographed and enlarged onphotographic paper. Gram-stained preparations of serialsections immediately adjacent to this section (two sectionson either side of the reference hematoxylin and eosin sec-tion) were examined with the light microscope. Every vesselthat contained a nocardial cell (either as a single cell or as anaggregate of bacteria) in any of these sections was marked onthe photograph.

Distribution of nocardiae within blood vessels. The type ofblood vessels in which the nocardiae adhered was deter-mined by a combination of light and electron microscopicanalysis. The nocardiae were not found in large vessels inthe brain, but they were localized in arterioles and capillar-ies. For this study, we used the following criteria to differ-entiate the type of blood vessel (13). (i) An arteriole wasdefined as a microvessel with a luminal diameter of less than100 p.m (usually about 40 ,um) with a wall that had no morethan one to two circularly arranged layers of smooth musclecells (arterioles tended to have a less distinct basal laminaand the endothelial cells were relatively thick). (ii) A capil-lary was defined as a microvessel that had a luminal diameterof less than 10 to 12 p.m with a single layer of relatively thinendothelial cells, a distinct basal lamina, and an occasionalpericyte surrounded by glial cells. (iii) A postcapillary venulewas defined as a microvessel that had a luminal diameter of10 to 30 ,um, a distinct basal lamina, an incomplete layer ofpericytes, endothelial cells of intermediate thickness (0.4 p.mthick) and that tended to collapse upon fixation.

RESULTS

Binding of N. asteroides GUH-2 to endothelial cells. Elec-tron microscopy showed that the nocardial cells bound to themembrane of endothelial cells of both arterioles and capil-laries of the pons and substantia nigra (Fig. 1). In someinstances, the outer membranous layer of the nocardial cellwall was bound firmly to the outer membrane of the endo-thelial cell adjacent to the tight junctions which formed theblood-brain barrier in the pons (Fig. 1A, arrow). Interactionssimilar to those observed in the pons were also seen in theregion of the substantia nigra (Fig. 1B). Figure 1B shows that

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A

FIG. 1. Electron micrographs of thin sections of the munine brain after perfusion with a suspension of single log-phase N. asteroidesGUH-2 cells showing the initial stages of nocardial attachment to the cytoplasmic membrane of capillary endothelial cells (arrows). (A)Capillary in the pons region of the brain. (B) Capillary in the substantia nigra region of the brain. Abbreviations: BM, basement membrane;ca, caveolae Lu, lumen of the capillary; TJ, tight junction.

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FIG. 2. Light micrograph of a coronal section of the murine braindemonstrating the sites for the binding of nocardiae after perfusionwith 7.5 x 107 CFU of log-phase N. asteroides GUH-2 cellsfollowed by a flush with saline to remove unattached bacteria. Eachdot represents either a single filament or groups of bacteria adherentwithin either arterioles or capillaries. This micrograph represents acomposite of four adjacent serial sections from a single representa-tive mouse (each 6.0 ,um thick). Similar regional sites of attachmentwere observed within the brains of duplicate mice as follows: 1,cerebral cortex; 2, thalamus; 3, hypothalamus; 4, substantia nigra;5, hippocampus (15).

the tip of the filamentous nocardial cell was bound to theendothelial cell membrane by way of electron-dense materialin the outer region of the nocardial cell wall (Fig. 1B, arrow).This attachment occurred rapidly (within 15 min) and ap-peared to be localized within certain regions of the brain(Fig. 2). The reason for perfusing the mouse with a largedose of nocardiae for 15 min (a total of approximately 7.5 x107 CFU per mouse) was to permit adequate opportunity forthe bacterial cells to bind to most of the attachment sites inthe brain. It was believed that this would facilitate visualiza-tion of the binding as well as show whether these attachmentsites were randomly distributed throughout the brain orlocalized within specific regions. It should be noted that thenocardial cells were localized in distinct areas of the brain,and they were not seen attached along all of the bloodvessels, even in the immediately adjacent areas (Fig. 2).

Alteration of the endothelial cell surface during attachment.Once the nocardial cell became firmly attached to theendothelial cell membrane, there was a deformation at thepoint of contact. As shown in Fig. 3A, the tip of thenocardial cell attached to the outer flap region adjacent to atight junction similar to that shown in Fig. 1A. The nocardialcell did not appear to be penetrating the tight junction (notethat the junction does not appear to be altered; Fig. 3A,arrow) but instead caused a collapse and compression of theflap region to form a cup around the tip of the nocardialfilament (Fig. 3A). Although Fig. 1A and 3A show nocardialcells close to the tight junctions of the endothelial cells in thepons region of the brain, it should be emphasized that themajority of the bacterial cells were not associated with thetight junction regions in the capillaries. One hundred differ-ent bacterial profiles were counted in both the pons and thesubstantia nigra regions. It was found that 13 of these cellsappeared to be next to the tight junction, whereas 87 werescattered along the endothelial cell surface (Fig. 3B). Severalendothelial cells had more than one bacterium attached percell. Figure 3B shows a nocardial filament attached to thesurface of an endothelial cell in an arteriole in the substantianigra region of the brain. As in Fig. 3A, the surface of theendothelial cell appeared to be forming an indentationaround the tip of the nocardial cell filament. The caveolae,pinocyte-like transport vesicles, from the surface of thebasement membrane appeared to be extending towards theattachment site on the endothelial cell membrane (Fig. 3B,arrow). This process was seen frequently, and it may repre-sent a possible mechanism whereby the nocardiae could passthrough the blood-brain barrier to enter the pericytes with-out disrupting the integrity of the blood-brain barrier.

Initiation of penetration of the endothelial cell. The nocar-diae appeared to attach mostly at the growing tip (apex) ofthe nocardial cell (Fig. 4; Table 1). Locci et al. (8, 9) haveshown that actinomycetes grow and elongate by apicalextension of the filament. At the growing site, the nocardialcytoplasm usually has a fine, granular zone, as shown in Fig.4A (Gz). Thus, this region of the nocardial cell must bemetabolically active because new cell wall is being formed atthis point. There appeared to be an active process initiated atthe point of attachment of the nocardiae to the endothelialcell (Fig. 4). This suggested that the growing nocardiaereleased a substance that affected the endothelial cell at thepoint of contact (Fig. 4). When 100 different bacterial cellprofiles were enumerated, it was found that 68 attached atthe tip of the filament as shown in Fig. 4A and B (Table 1).Only 32% of the nocardiae were attached along the side ofthe filament, and none of these showed evidence of penetra-tion. Furthermore, 70.8% of the cells that attached at theirapexes appeared to penetrate the endothelium (Table 1). Thebacterial cell shown in Fig. 4C had attachment along thefilament, but penetration appeared to occur only where aside branch of the cell was developing (the growing tip; Fig.4C, arrow).

Penetration of the endothelial cell. Figure 5 shows that oncethe nocardial filament attached to the surface of the endo-thelial cell, a rapid response was induced. This process hadto occur within a time frame of a minimum of 10 min to amaximum of 25 min. This attachment and penetration musthave had significant rigidity because the filamentous cellshown in Fig. 5 was at right angles to the cell surface againstthe flow of fluid through the blood vessel (Fig. SA). Figure5B is a high magnification of the serial section of the cellshown in Fig. SA. Note that there was an increased numberof caveolae at the basement membrane interacting with the

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FIG. 3. Electron micrographs of thin sections of the perfused murine brain (as in Fig. 1) showing attachment of N. asteroides cells at thefilament apex resulting in an altered endothelial cell surface (formation of a cuplike deformation). (A) Pons region (10 to 25 min afterexposure). (B) Substantia nigra region (10 to 25 min after exposure). Arrows indicate tight association between nocardial cell wall andendothelial cell membrane. Abbreviations: TJ, tight junction; Lu, lumen of the capillary; ca, caveolae.

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FIG. 4. Electron micrographs of thin sections of the perfused murine brain (as in Fig. 1 and 2) showing the early initiation of penetrationof the endothelial cells by the nocardiae. (A) Substantia nigra region (10 to 25 min after exposure). (B) An arteriole in the pons region (10 to25 min after exposure). (C) A capillary in the pons region (10 to 25 min after exposure). Abbreviations: Gz, granular zone frequently seen atthe growing tip of the nocardial filament; N, endothelial cell nucleus; ca, caveolae; BM, basement membrane; Lu, lumen of blood vessel; M,mesosome of nocardial cell. Arrows point out tight association between nocardial cell wall and endothelial cell membrane.

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TABLE 1. Quantitation of nocardial attachment and penetrationin brain stem and midbrain, thalamus, and hypothalamus inBALB/c mice 15 to 25 min after intra-arterial perfusiona

Adherence (%)bNocardiae Penetration (%)C

Apical Axial

Live cells 68 32 70.8Heated-killed cells 52 48 0

a The mice were perfused with 7.5 x 107 CFU of live, log-phase cells orwith approximately the same cell density of heat-killed cells.

b One hundred random bacterial cell profiles were counted (serial sectionsshowing the same cells were excluded). The same results were obtained whenthese experiments were repeated.

c Penetration was quantitated by scanning electron microscopy. One hun-dred bacterial cells were counted; only cells attached by the apex penetratedthe endothelial cell; therefore, the values represent percent apical attachment(axially adherent cells were excluded).

endothelial cell membrane (Fig. 5B, "ca" arrow). Therewere also several points of contact between the bacterial cellwall and the endothelial membrane in the penetrating vesicle(Fig. SB, arrows). This process appeared to be due to anactive alteration of the endothelial cell and not due tonocardial extension by growth, because of the amount oftime involved. (It should be noted that within a 25-minincubation period little elongation of the nocardial filamentcan occur because the cellular doubling time under idealconditions for this organism is at least 3 h, with an initial lagphase of 6 to 8 h in vivo [4, 5].)The penetration of the endothelial cell by the nocardiae, as

shown in Fig. 5, suggested that there was an inducedphagocytic response in the endothelial cell. However, scan-ning electron microscopy of the surface interactions betweennocardial cells and the endothelium did not support thisinterpretation (Fig. 6A and B). Even though there may besome induction of phagocytosis, the nocardiae appeared toactively penetrate the endothelial surface (Fig. 6A and B,arrows). Furthermore, as shown by thin sections, scanningelectron microscopy demonstrated that the nocardiae whichattached along the side of the filament (axial attachment) didnot penetrate the endothelium (Fig. 6C, arrow). This pene-tration of the endothelium appeared to be an active processinduced by some substance at the growing tip of the cell,because heat-killed cells still attached by the apex to theendothelial surface but did not penetrate (Fig. 6D; Table 1).

Internalization of nocardiae into the endothelial cell. Elec-tron microscopic analysis clearly demonstrated that thenocardiae were internalized within endothelial cells (Fig. 7Ato E). These bacteria extended into both the cytoplasm (Fig.7D and E) and the nucleus (Fig. 7F) within 10 to 25 min aftercontact with the endothelial cell. During this time, there waslittle or no evidence of damage to the host cell. Furthermore,the bacteria remained enclosed by a membrane (Fig. 7D, E,and F, arrows), and there was no evidence of damage to thebacterial cellular integrity (Fig. 7).

DISCUSSIONThe light and electron microscopic observations presented

above, combined with viable count determinations aftermicrodissection of regions of the brain (11, 12), indicatedthat there was a cell surface receptor for the binding of N.asteroides GUH-2 to endothelial cells in arterioles andcapillaries localized within the pons, midbrain, thalamus,and hypothalamus. This receptor recognized a componenton the outer surface of the bacterial cell wall, closely

associated with the growing end of the nocardial filament.Furthermore, the binding of the nocardiae to the membraneof the endothelial cell occurred within a 15-min exposureperiod, and this binding was not preferentially associatedwith the endothelial intercellular junction (tight junction).Indeed, none of the nocardial cells enumerated (0 of 100)were penetrating the blood vessel through the tight junctionswhich remained intact. The level of this attachment wasreduced in frequency, but it was not eliminated by killing thenocardiae at 60°C.At the site of attachment, the nocardial cells began to

enter the endothelial cell by a process that appeared to be anactive penetration because dead cells did not penetrate theendothelium. Often this process occurred in the nuclearregion, whereby the nucleus became displaced or indented,and nocardial cells were found in both the cytoplasm and thenucleus of the endothelial cell. During this internalizationprocess, all membranes (both cytoplasmic and nuclear) wereintact, and the bacterial cell remained within a membrane-bound vesicle.There are few reports on the ultrastructural analysis of the

interactions of other bacteria with intact capillary endothe-lial cells in the brain. However, there have been numerousstudies on microbial interactions with in vitro-cultured en-dothelial cells obtained from the arteries or veins from avariety of hosts (6, 10, 14, 18, 19). Rickettsiae were shown tobind to the surface of cultured endothelial cells, wherebythey facilitated an active phagocytosis (19). It was shownthat killed rickettsiae could still bind to the cell surface, butthey were not phagocytosed unless they were metabolicallyactive (19). Furthermore, treatment of the endothelial cellswith agents that prevented phagocytosis also preventeduptake of the rickettsiae (19). Therefore, with rickettsiae,attachment to endothelial cells appears to be passive andindependent of the interactive process of facilitated phago-cytosis (19).

Initially it was believed that the nocardiae might beattaching to the endothelial cell surface and inducing aphagocytic response, as seen with the rickettsiae (19). How-ever, scanning electron microscopic analysis of this processrevealed that the nocardiae were actively penetratingthrough the surface with displacement of the cytoplasmicmembrane of the endothelial cell. These data suggest thatgrowing N. asteroides GUH-2 cells produce an invasionfactor that is either heat labile or secreted only by the livecells.Even though endothelial cells are not generally regarded

as primary phagocytic cells, it has been established thatthese cells can be induced to phagocytose particles in amanner similar to the professional phagocytes (14). It wasshown that Borrelia burgdorferi cells bind to the surface ofhuman umbilical vein endothelial cells in vitro, and they areinternalized by a phagocytic process (6). Furthermore, thesespirochetes attach to endothelial cell monolayers and thenpenetrate through the intercellular tight junctions (16).Therefore, it is thought that B. burgdorferi invades the brainduring neuroborreliosis by a specific attachment to theendothelial cell followed by penetration of the blood-brainbarrier through the tight junctions (16). This is in sharpcontrast to the data presented above, which indicate thatnocardial cells have a specific binding to a subpopulation ofendothelial cells because the nocardiae are not bound to allblood vessels throughout the brain. After attachment, thereis rapid penetration of the endothelium by the nocardiae.Furthermore, the nocardial cells do not appear to penetratethrough the blood-brain barrier at the intercellular tight

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FIG. 5. Nocardial penetration of an endothelial cell in a capillary in the pons region of the brain. (A) Tangential section of the capillaryshowing right angle penetration of the endothelial cell by a nocardial filament at 10 to 25 min after exposure. The double arrow indicates thedirectional flow of fluid through the capillary. Bar = 1.0 ,um. (B) High-magnification insert of a serial section of the same cell shown in panelA. Note the tight contacts between the nocardial cell wall (arrows) and endothelial cell membrane in what appears to be a phagocytic processby the host cell. Note the increased numbers of caveolae (ca) interacting between the phagosomal membrane and the basement membrane(BM) and the apparent dissolution of the basement substance in the area of contact (curved arrows).

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FIG. 6. Scanning electron micrographs of the attachment and penetration of arterioles and capillaries by log-phase N. asteroides GUH-2cells within regions of the midbrain, thalamus, and hypothalamus. (A) Penetration (arrows) of GUH-2 cells (live) through the surface of theendothelium of an arteriole in the thalamus. Note the nocardial cell adherent in and protruding from a capillary as well as several nocardiaeadhering axially at the bifurcation. (B) High magnification view of two nocardial cells (live) penetrating through the endothelium of an arteriolein the region of the hypothalamus (arrows). (C) A live nocardial cell attached axially near the tip of the filament (arrow). Note the failure todeform or penetrate the surface of the endothelium at the site of attachment (capillary in the midbrain). (D) Attachment of dead (heat-killed)nocardiae in a capillary in the midbrain near the region of the substantia nigra. Note the attachment of the tips of the nocardial filament atthe surface of the endothelium with no evidence of penetration (arrows). Bars = 1 p.m.

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FIG. 7. Electron micrographs of thin sections of different nocardiae at various stages of internalization into endothelial cells. (A) Activepenetration of nocardial filament into an endothelial cell in a capillary in the substantia nigra region (10 to 25 min after exposure). Note theindentation of the nucleus (N). (B) A process similar to that in panel A, only in a capillary in the pons region (note the displacement of thenucleus). (C) A nocardial cell that is in the final stage of becoming internalized by what appears to be an actively phagocytic process (arrows)in the substantia nigra region (10 to 25 min after exposure). (D) A nocardial cell within a phagosome (arrow) in the cytoplasm of a capillaryendothelial cell (note the adherence of the phagosome to the basement membrane [curved arrow]). Note that there are several bacterial cellswithin this region of the capillary. (E) A nocardial cell in a phagosome in the cytoplasm of an endothelial cell. Note that the organism is locatedbetween the nucleus and basement membrane (BM) (10 to 25 min after exposure). (F) A nocardial cell within a phagosome (arrow) lyingdeeply within the nucleus of a capillary endothelial cell in the substantia nigra region (10 to 25 min after exposure). Bars = 1 p.m.

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NOCARDIAL ATTACHMENT TO AND PENETRATION OF CAPILLARIES 965

junction, but instead, these bacteria may be transportedthrough the endothelial cell to the basement membrane in amanner analogous to the transport of Shigella organismsthrough intestinal columnar epithelial cells to the laminapropria (10, 17, 18). If a similar transport mechanism occursfor the nocardiae, then this process appears to be facilitatedby an invasion factor.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant R01-AI20900 from the National Institute of Allergy and InfectiousDiseases.We thank Robert Munn, Department of Pathology, Electron

Microscope facility, for his help in pathologic interpretation of someof the electron micrographs during this study. We also thank LynnDiaz and Marilyn Kiene for typing the manuscript.

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3. Beaman, B. L., M. E. Gershwin, S. S. Scates, and Y. Ohsugi.1980. Immunobiology of germfree mice infected with Nocardiaasteroides. Infect. Immun. 29:733-743.

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