Microbial Pathogenesis 1991 ; 10 : 231-245

Interactions of Haemophilus influenzae with cultured human endothelial cells Mumtaz Virji,'* Helena Kayhty,'t David J . P . Ferguson, 2 Carol Alexandrescu' and E . Richard Moxon' 'Molecular Infectious Diseases Group, Department of Paediatrics, 2 Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, U.K. (Received November 15, 1990 ; accepted in revised form December 19, 1990)

Virji, M . (Molecular Infectious Diseases Group, Dept of Paediatrics, John Radcliffe Hospital, Oxford, U .K .), H . Kayhty, D . J . P . Ferguson, C . Alexandrescu and E . R . Moxon . Interactions of Haemophilus influenzae with cultured human endothelial cells . Microbial Pathogenesis 1991 ; 10:231-245 . The interactions of capsulate (b`) and capsule-deficient (b - ) Haemophilus influenzae type b with endothelial cells were studied in vitro using human umbilical vein endothelial cells (Huvecs) . Association was determined by estimation of colony forming units (cfu) as well as the binding of 3 H-thymidine-labelled bacteria . Bacteria associated with Huvecs rapidly and in a dose-dependent manner. The presence of capsule on bacteria resulted in a decrease in the rate of cell-association . Internationalisation of bacteria by Huvecs was quantitated after elimination of extracellular bacteria with gentamicin . It was found that larger numbers of b bacteria were internalised compared to b' bacteria . Incubation in the presence of metabolic inhibitors had little effect on the association of bacteria to Huvecs whereas internalisation was dependent on the integrity of host cellular functions . Electron microscopic studies confirmed phagocytic ingestion of both b' and b - variants and suggested that the majority of the internalised bacteria remained viable within endothelial cell vacuoles . Haemophilus influenzae were translocated within vacuoles both from the apical to basal and the basal to apical direction . Key words : Haemophilus influenzae ; capsule ; endothelial cell ; association ; internalisation .

Introduction

Haemophilus influenzae type b is a major cause of bacteraemic infections such as meningitis . The mechanisms which enable the bacterium to disseminate to blood and the brain are imperfectly understood . Possible routes of entry into the bloodstream from the nasopharynx, the site of initial colonisation,' include direct invasion of the submucosal blood vessels or transfer to regional lymph nodes and subsequently via the efferent lymphatics to the blood . These events might involve free bacteria or organisms associated with phagocytes . Previous studies using the infant rat model of meningitis indicated that early transient bacteraemia resulted from direct invasion of blood vessels in the sub-epithelial tissues of the nasopharynx .' In addition, since meningitis is preceded by bacteraemia, 3 establishment of CNS infection might involve direct interaction of H . influenzae with the endothelial cells in the breaching of the Author to whom all correspondence should be addressed at : Oxford University Department of Paediatrics, John Radcliffe Hospital, Oxford 0X3 9DU, U .K . t Present address : National Public Health Institute, Helsinki, Finland . 0882-4010/91/030231 +15 $03 .00/0

© 1991 Academic Press Limited



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No . present (log, o ) Fig . 1 . The effect of bacterial concentration on association of Rd b - ( D) and Eag b' ( •) with Huvecs 3 h post-inoculation . Results are the mean of triplicate estimations (SDs 5 ±25% of the mean) . When the inoculum was below 10 9 bacteria per monolayer, linear relationships were observed whose correlation coefficients were 0 .91 (Rd b- ) and 0.9 (Eag b') .

blood-brain barrier. In order to investigate the interaction of H. influenzae with endothelial cells, we have used human umbilical vein endothelial cells (Huvecs) in vitro to study the association, internalisation and translocation of two strains of H. influenzae, Eagan (Eag) and Rd . These studies have also compared isogenic variants of both strains either expressing (b+) or lacking (b - ) the ribosyl-ribitol phosphate capsule in order to analyse the effect of this capsule on bacterial interactions with the endothelial cells . Results Quantitative studies Association of H . influenzae with Huvecs . Association of both b + and b - variants was related to the number of bacteria present over a range of concentrations (Fig . 1) . In the case of b - organisms, saturation of association was observed within 3 h when > 109 bacteria were present per monolayer of 3 x 104 H uvecs . Kinetics of association . To study the rate of association with Huvecs, an inoculum of 5 x 10 8 bacteria per monolayer was used since, at this density and under these incubation conditions, no bacterial growth occurred and the numbers of viable bacteria remained constant for the 6 h duration of this experiment . Initially cell association of both b+ and b - variants increased rapidly, but by 30 min relatively larger numbers of capsule-deficient bacteria had associated with Huvecs as compared with the capsulate variants (Fig . 2) . The difference between numbers of cell-associated b+ and b progressively diminished over longer incubations (Figs 2 and 3) . Comparison of the association of b+ and b - H. influenzae with four different Huvec preparations (passage 1) was carried out simultaneously to assess the reproducibility of the findings . The total numbers of cell-associated bacteria varied between different



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Time (h) Fig . 2 . Association ( ) and internalisation ( ) of H. influenzae as a function of time . (a) Eag b (o), Eag b' (t) ; (b) Rd b - (n), Rd b' (A) . The absolute numbers of bacteria remained constant during the experiment . Each point represents a mean of at least three values (SDs < ±25% of the mean) .

Huvec preparations (up to five-fold difference) . When examined within first few hours of incubation, b variants consistently associated with each Huvec preparation in larger numbers compared with b+ variants . Figure 2 summarises the relative differences between b + and b H. influenzae . Internalisation . To investigate the internalisation of H. influenzae by Huvecs, extracellular bacteria were eliminated with gentamicin . Figure 2 shows that the b organisms were internalised more rapidly than b + organisms, results which reflected the different patterns of cell-association of the capsule-deficient and capsulate bacteria . In addition, as with association, the difference between numbers of internalised b' and b - decreased over longer incubations (Figs 2 and 3) . Figure 3 also shows that intracellular numbers of both b + and b - bacteria reached a steady state and remained constant over several hours . Metabolic requirements . To investigate whether association or internalisation was dependent on active cellular mechanisms, metabolic functions of bacteria and Huvecs were modified by the use of several inhibitors (Tables 1 and 2) . Association was not significantly reduced by inhibition of Huvec metabolic activity (4°C, dinitrophenol, fixation) or by inhibition of microfilament function (cytochalasin D) . Indeed, reduction in surface activity resulted in increased association of bacteria with Huvecs in some cases (Table 1) . Similar observations have been reported by other workers . 4 In contrast



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Fig . 3 . Mean±SD numbers of bacteria associated with Huvecs (o) and intracellular bacteria (®) and 20 h after inoculation with 2 x 10 6 bacteria per monolayer .

Table 1 Effect of temperature, cytochalasin D and fixation on association and internalisation of H. influenzae strain Eagan by Huvecs Huvec treatment None 4°C Cytochalasin D Paraformaldehyde fixation

Association of Eag b'

Internalisation of Eag b"

100 137 115 80

100 2 3 0 .1

Association and internalisation of Eag b' was determined 3 h after incubation with treated and untreated Huvecs . Experiments were done in triplicate . All results are expressed as percentages of the untreated controls .

Table 2 Effect of tetracyclin, gentamicin and dinitrophenol on association of H. influenzae strain Rd with Huvecs Percent association Treatment None Tetracyc l i n Gentamicin Dinitrophenol

Rd b

Rd b -

100 119 94 79

100 118 115 84

Association of bacteria with Huvecs under bacteriostatic and bactericidal conditions was determined after 3 h incubation . All experiments were done in triplicate . Results are expressed as percentages of the untreated controls .



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and in spite of the presence of large numbers of bacteria on the surface, inhibition of Huvec metabolic functions and phagocytic activity resulted in a significant reduction in the numbers of internalised bacteria (Table 1) . Treatment of bacteria with tetracyclin, dinitrophenol or gentamicin had little effect on their association with Huvecs (Table 2) . These data suggest that adherence of bacteria to endothelial cells does not require de novo synthesis of either host or bacterial adhesive components, observations which are consistent with the rapid kinetics of association shown in Fig . 2 . However, for each of the variants, internalisation of bacteria into Huvecs was dependent on intact microfilament functions . Effect of bacteria on Huvec mono/avers . Effect of H . influenzae on Huvec monolayer integrity was investigated using light microscopy . Another mucosal pathogen, Neisseria meningitidis, was also included in these studies for comparison . It was found that while N. meningitidis caused gross disruption of the monolayers, H. influenzae had no apparent effect and host cells were viable 18 h after infection as indicated by trypan blue exclusion . Bacterial numbers had increased to over 5x10 8/monolayer by the end of the 18 h incubation in each case (Fig . 4) . Ultrastructural observations Using scanning and transmission electron microscopy (SEM and TEM) Huvec monolayers were found to present smooth apical surfaces with a few small microvilli [Fig . 5(a)] . Cross-sections through the monolayers showed that the cells were flattened and there was some overlap of adjacent cells which were connected by tight junctions [Fig . 6(a) and (h)] . The presence of Weibel-Palade bodies, the characteristic rod-shaped cytoplasmic inclusions found in normal endothelial cells,' was observed in several preparations . Examination of monolayers fixed after 30 min incubation showed bacteria attached to the surface of Huvecs . It was consistently observed that large numbers of b compared with b' organisms adhered to Huvecs . In addition, there were marked

Fig . 4 . Photomicrographs showing the effect of bacteria on Huvec monolayers . Huvecs were inoculated with 2x106 bacteria per monolayer . (a) Haemophilus influenzae strain Eag b ; (b) Neisseria meningitidis strain MC58 . Monolayers were washed after 18 h exposure and stained with giemsa .



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Fig . 5 . Scanning electron micrographs of Huvecs grown on coverslips and exposed to either b' or b variants of H. influenzae . ( a) Lower power micrograph showing the relatively smooth surface of the endothelial cells and large numbers of Rd b - bacteria (B) associated with one endothelial cell with few or none on adjacent cells . Bar represents 10 pm . (b) Detail showing two Rd b - bacteria (B) partially covered by a cytoplasmic process from a Huvec (arrow) . Bar represents 0.5 pm . (c) A bacterium (Eag b') partially enclosed by a cytoplasmic fold of an endothelial cell (arrow) . Bar represents 0.5 pm . (d) Surface of a Huvec showing two adherent Rd b - bacteria (B) plus one almost enclosed within the endothelial cell (arrow) . Bar represents 0 .5 pm . (e) Surface of a Huvec showing a number of bacteria (Eag b') apparently located under the plasmalemma of the endothelial cell (arrows) . Bar represents 0 .5 pm .



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differences in the numbers of bacteria attached to different cells within the same monolayer [Fig . 5(a)] . The bacteria appeared randomly distributed over the surface with no specific localisation at the cell junctions [Fig . 5(a)] . This variation in the number of attached b+ and b - bacteria was confirmed by TEM which also provided morphological evidence to support the endothelial nature of the cells in spite of observed differences in attachment . Quantitation of organisms adherent and internalised after 5 h incubation with Huvecs also showed that the largest number of adherent bacteria correlated with the largest number of intracellular bacteria . Internalisation . The SEM and TEM studies suggested a possible sequence of events associated with bacterial internalisation . This process was similar for both the b + and b H. influenzae. Cytoplasmic processes partially enclosing the bacteria were observed [Fig . 5(b)] . These formed cup-like structures [Fig . 6(b)] which progressed until the bacterium was enclosed [Fig . 5(c) and (d)] . In certain cases, the cytoplasmic folds appear to coil around the bacterium [Fig . 6(c)] in a manner previously described for internalisation of Legione/la pneumophila by monocytes .' Close apposition of the overlapping cytoplasmic processes was often seen enclosing the bacterium within the endothelial cell [Fig . 6(d)] presumably followed by fusion resulting in internalisation of a bacterium within a membrane-bound vacuole in the endothelial cell cytoplasm [Fig . 6(e)] . This intracellular location was confirmed by ruthenium red staining since the latter is known to be excluded from intracellular vacuoles [Fig . 6(f)] . The location of bacteria below the plasma membrane was also observed by SEM [Fig . 5(e)] . Intracellular fate . The intracellular bacteria were always situated within membrane bound vacuoles [Fig 6(e)] . The majority of vacuoles contained single organisms but vacuoles with multiple bacteria were also observed [Fig . 6(a)] . Although the latter could have resulted from internalisation of microcolonies or aggregated bacteria, the possibility of intracellular growth was not excluded . In addition, a variable number of fine cytoplasmic filaments (possibly actin) accumulated around some vacuoles which contained bacteria [Fig . 6(e)] . Similarly, masses of filaments were also observed in certain cells during bacterial internalisation [Fig . 7(c)] . The majority of intracellular bacteria exhibited normal morphology [Fig . 6(e)] although occasional vacuoles that contained degenerate bacteria were observed . The bacteria were distributed throughout the endothelial cell cytoplasm . In several cases, there appeared to be fusion of the vacuolar membrane with the basal plasmalemma [Fig . 6(g)], a finding which was consistent with the presence of bacteria located beneath the intact Huvec monolayer [Fig . 6(h)] . Interaction with basal endothelial cell surface . An unexpected but consistent finding was that when Huvecs were seeded on 3 .0-/cm pore filters confluent monolayers formed on both sides of the filters . This occurred whether they were seeded at or below confluency and even if only the top surface had been treated with gelatin . By infecting from above only, it was possible to use these monolayers to study not only the apical to basal translocation of bacteria at the upper monolayer (described above) but also the basal to apical translocation at the lower monolayer . Transmission electron microscopic examination of these filters showed that some bacteria had penetrated the upper monolayer within 3 .5 h and were located within the pores of the filters . Furthermore, bacteria were also seen at the basal surface and within the lower monolayer [Fig . 7(a)] . Stages in bacterial internalisation at the basal surface of the endothelial cells could be identified and were similar to those observed at the apical surface [Fig . 7(b) and (c)] . The resulting intracellular bacteria were situated within vacuoles which, in certain cases, appeared close to the apical surface of the endothelial cell [Fig . 7(d)] . This may represent basal to apical translocation, the reverse of that seen in the upper monolayer .

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(a)

(b)

(c)

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It should be noted that only a small number of bacteria were present at the apical surface of the lower monolayer ( < 5% of that at the basal surface) . Thus internalisation at the apical surface of the lower monolayer was infrequent . Intercellular location . I n addition to the intracellular location, bacteria were sometimes observed between endothelial cells on both the upper [Fig . 7(e)] and lower [Fig . 7(f)] monolayers . The bacteria were often located between overlapping cytoplasmic processes of the endothelial cells in areas where the cell junctions were maintained [Fig . 7(f)] . Occasionally, however, small gaps between endothelial cells were observed and bacteria were found near these gaps .

Discussion In the present studies, we used two genetically distinct strains of H. influenzae to evaluate the role of the ribosyl-ribitol phosphate capsule in bacterial interactions with endothelial cells . One consistent finding was that capsule-deficient bacteria associated with and were internalised by endothelial cells more rapidly than the capsulate derivatives . One possible mechanism by which the presence of capsule on a bacterium might interfere with its interactions with host cell surface is the introduction of a charge barrier due to negatively charged ribosyl-ribitol phosphate capsule and endothelial cell surface .' Another possibility is that the capsule might mask or sterically hinder any specific adhesin/s that might be present on the bacterial surface . Pilus and non-pilus adhesins which may facilitate H . influenzae adhesion to epithelial cells have been described .'-" The strains used in the present studies however, did not express pili (as confirmed by lack of haemagglutination and by electron microscopy) . Whether the two strains possess specific non-pilus adhesin/s or indeed whether the same or different adhesins mediate attachment of the two strains to Huvecs requires further investigation . Our data show that the observed interactions did not require de novo synthesis of host or bacterial components . However, the possibility remains that growth of bacteria under other environmental conditions may alter association or internalisation by alteration in surface composition ." Two features were noted in the interactions of H. influenzae strains with different Huvecs . Quantitative differences in the association were observed between preparations of Huvecs from different cords . In addition, some cells of the same origin were able to bind more bacteria than others . Whether these differences are linked to genetically determined differences in expression of host antigens is not known . Differences between cells of the same origin may be due to variation in the state of Fig . 6 . Transmission electron micrographs of cross-sections through Huvecs on the upper surface of 0 .4and 3-dim filters exposed to either b' or b - variants of H. influenzae . All micrographs are orientated with the Huvec apical surface upwards . (a) Low power micrograph showing part of the Huvec monolayer with a number of extracellular Rd b - bacteria (B) and large number of bacteria located both singly and in groups within the endothelial cell cytoplasm (arrowheads) . Bar represents 2 pm . (b) Detail showing a bacterium (B) associated with cup-like folds of the cytoplasm of an endothelial cell . (Rd b' variant) . Bar represents 0 .5 pm . (c) A cytoplasmic fold from a Huvec is shown coiled around a Rd b bacterium (arrow) . Bar represents 0 .5 pm . (d) Bacterium (B) enclosed by two closely adherent overlapping cytoplasmic processes of the Huvec (arrowhead) . Rd b' variant . Bar represents 0.5 pm . (e) Two bacteria located within vacuoles (V) in the endothelial cell cytoplasm are shown . Note the variable number of fine cytoplasmic filaments associated with these vacuoles (arrows) . (Rd b - variant) . Bar represents 0 .5 pm . (f) Sample treated with ruthenium red showing the electron dense staining of the glycocalyx of the apical plasmalemma of the Huvec (arrowheads) and the absence of staining on the vacuolar membrane enclosing the bacterium (arrows) consistent with its intracellular location . (Rd b - variant) . Bar represents 0.5 /rm . (g) The apparent fusion of the membrane of a phagocytic vacuole with the basal plasmalemma of an endothelial cell is shown . (B : Rd b variant) . Bar represents 0 .5 pm . (h) A number of Rd b - bacteria (B) located beneath the endothelial cells is shown . Note the intact junction between endothelial cells (arrow) . F : filter . Bar represents 0 .5 ppm .



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(a)

Fig . 7 . Transmission electron micrographs of cross-sections through endothelial cells on both sides of 3 pm filters exposed to Rd b - variant from the upper surface only . Figures (b)-(d) and (f) are from the lower surface of the filter and are thus orientated with the apical surface of the Huvecs facing downwards . Figure (e) is from the upper monolayer with the apical surface upwards, (a) Montage showing intact Huvec monolayers on both sides of the filter (F) . Note the bacteria (B) above, within and under the upper Huvec, within the filter pores, and within the lower Huvec . Bar represents 5 pm . (b) Detail showing bacterium (B) associated with a cytoplasmic process from the basal surface of an endothelial cell . F : filter . Bar represents 0 .5 pm . (c) Bacterium (B) entering an endothelial cell via the basal surface . Note the accumulation of fine filaments in the Huvec cytoplasm around the point of entry (arrowhead) . Bar represents 0 .5 pm . (d) Enlargement of the enclosed area in (a) showing a bacterium (B) located with a vacuole close to the apical surface of an endothelial cell . Bar represents 0 .5 dim . (e) Bacteria located between overlapping endothelial cells of the upper monolayer are illustrated (arrows) . Bar represents 0 .5 pm . (f) Detail of lower monolayer showing two bacteria (arrows) located between endothelial cells which have retained their junctions (arrowheads) . Bar represents 0 .5 pm.



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differentiation of the individual cells . For example, variable expression of surface antigens e .g . blood group antigens in a clonal population of endothelial cells has been 3,14 described .' Bacteria were internalised by Huvecs by a phagocytic process which required cytoskeletal activity . Other workers have reported uptake of different bacterial strains by endothelial cells ."- " Haemophilus influenzae can interact and appear to be internalised both at apical and basal surfaces of the polarised cells . This could have important implications in pathogenesis . Bacteria may enter the blood by direct invasion of pharyngeal vessels (basal to apical translocation) .' However, once bacteraemia is established, apical to basal translocation may be involved in central nervous system entry . We followed the fate of intracellular bacteria by a quantitative assay using gentamicin to eliminate extracellular bacteria and by examination in TEM . Quantitative assays showed that intracellular numbers of both b' and b increased until a steady state was reached when the numbers inside remained constant over several hours . Intracellular numbers of bacteria represent an equilibrium between the processes of multiplication, death, internalisation and exocytosis . Examination of a large number of electron micrographs failed to show evidence of intracellular multiplication or destruction . Further, bacterial numbers did not decrease on incubation with Huvecs over a wide range of inocula . Apparently therefore, under the conditions used there was little destruction of H. influenzae ; instead a consistent increase of bacterial numbers was observed in the presence of Huvecs . It was interesting to note that despite the large numbers of bacteria present during incubations with Huvecs (in some experiments for up to 20 h), these appeared intact by light microscopy and cells in the monolayers were viable as indicated by trypan blue exclusion . Electron microscopic examination of monolayers exposed to bacteria for 6-7 h showed that a large number of bacteria had penetrated the monolayer without overall destruction of the monolayer or changes in Huvec morphology . This is in sharp contrast with the effect of another mucosal pathogen, N . meningitidis which, in comparable experiments, caused extensive destruction of the monolayer ." Endothelial cells occupy an important strategic location within the body and the nature of their interactions with pathogenic microbes could be expected to influence the outcome of an infection . To cross this barrier, an organism may use one of several routes . Direct routes may involve passage between cells as has been described for motile organisms such as Treponema pallidum 20 or intracellular translocation .' $ Since endothelial cells exhibit macrophage-like phagocytic properties," this route may be potentially utilised by any bacterium able to overcome or to escape intracellular bactericidal mechanisms which although present, are relatively inefficient at killing phagocytosed bacteria ." Consequently one could speculate that some bacteria may utilise the location within the endothelial cells as a privileged site to escape the professional phagocytes and humoral defences . Such a model does not necessitate intracellular growth, merely the ability of a bacterium to localise within the host cells and to exit either by exocytosis or on cell death . Our data show that H . influenzae can enter endothelial cells, remain localised within membrane-bound vacuoles, traverse the depth of the cell and appear to emerge at the opposite surface . Since both b - and b H. influenzae appear to translocate, the greater virulence of the capsulate bacteria cannot be explained on the basis of their ability to cross the endothelial barrier . Materials and methods Bacteria and growth conditions . Capsulate and non-capsulate isogenic derivatives of two strains of H. influenzae were used in these studies . Haemophilus influenzae type b strain Eagan



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is a clinical isolate and produces meningitis in the infant rat model, 22 Two isogenic noncapsulate derivatives of this strain have been described previously . 23,24 RM804 is a spontaneous capsule-deficient mutant and RM248 is a deletion mutant of strain Eagan . Comparison of the two capsule-deficient derivatives in preliminary experiments (association and uptake described below) showed that they behaved identically and therefore only RM248 was used for subsequent experiments . Strain Rd - /b' :02 (RM135) and its spontaneous capsule-deficient mutant (Rd-/b- :02, RM132) have been described previously . 25 In addition, RM792 was obtained by transformation of RM132 with cloned DNA derived from strain Eagan . 26 RM132 is relatively avirulent whereas RM135 and RM792 caused bacteraemia and meningitis in the infant rat model .25 .26 In comparative studies the capsulate derivatives RM792 and RM135 behaved similarly and so only RM792 was used . The presence of capsule on bacteria used in the experiments was confirmed by their iridescence 23 and agglutination by specific antisera against type b capsule (Wellcome) . All variants employed in these studies were non-piliated since they failed to agglutinate human erythrocytes ." The absence of pili was also confirmed by electron microscopy . For clarity, the following abbreviations are used in the text : Eagan : Eag b', RM248 : Eag b ; RM792 : Rd b', RM132 : Rd b - . All strains were maintained at -70°C and grown on brain heart infusion (BHI) agar (Difco) supplemented with 10% Levinthal base or in BHI broth supplemented with 2 µg/ml NAD and 10 pg/ml hemin . Endothelial cells . Endothelial cells from human umbilical cords were obtained essentially as described by Jaffe ." Briefly, endothelial cells were released from the cords by incubation for 10 min with 0 .05% collagenase solution (Boehringer) . Cells were washed, suspended in growth medium (GM) containing antibiotics (see below), seeded in a 25 cm 2 tissue culture flask and incubated until confluent (3-4 days) . For a given experiment, cells from passages 1-4 were seeded in appropriate tissue culture plates and used 3-4 days after reaching confluency . All flasks and plates used for culture of Huvecs were pre-coated with 0 .2% gelatin (Sigma) overnight at 37°C . The endothelial nature of the cells was confirmed by their typical 'cobblestone' appearance at confluency, and factor VIII antigen expression by immunostaining using rabbit anti-factor VIII antibody (Dako) and goat anti-rabbit antibody conjugated to fluorescein (Dako) . The presence of Weibel-Palade bodies was often detected by electron microscopy .' Media . Growth medium (GM) : Huvecs were grown in bicarbonate buffered medium 199 with Earl's salts (Gibco) supplemented with 25% heat inactivated (56°C, 30 min) foetal bovine serum (FBS), 40 pg/ml endothelial growth supplement (Sigma), 90 pg/ml heparin (Sigma), 3 mm glutamine, 100 pg/ml streptomycin and 100 units/ml penicillin . Two to 24 h before use in infection experiments, Huvecs were washed twice with Hanks' balanced salts (HBSS, Gibco) supplemented with 2% FBS (HBSS-FBS) and further incubated in maintenance medium (MM) : medium 199 containing 15% FBS in addition to heparin and glutamine as above .

Determination of bacterial association with endothelial cells Viable count method. Endothelial cells were seeded in 96-well tissue culture plates in 100 µl GM which was replaced with antibiotic-free MM as above . Strains of H. influenzae were inoculated onto agar plates from stocks frozen at -70°C . Eighteen to 20 h cultures were suspended in Dulbecco's complete phosphate buffered saline (PBSB, Sigma) and spun at 85xg for 1 min to remove any large aggregates . Bacterial numbers were estimated by solubilisation of aliquots in 1% SDS in 0 .1 M NaOH and by measuring absorbance at 260 nm . Each suspension was diluted in MM to the required density . Fifty microlitres of medium was removed from microtitre-wells containing endothelial cultures and 50 µl bacterial suspension was added . Plates were incubated for desired intervals at 37°C in a CO 2 incubator . Samples (10 pl) were removed from each well for the estimation of bacterial numbers at the end of incubation . Monolayers were then carefully washed four times with HBSS-FBS. Further washing did not significantly reduce the numbers of associated bacteria . Forty microlitres of 1 % saponin in PBSB containing 2% FBS was then added to the wells to release associated bacteria . Dilutions were prepared within 10-20 min and plated on agar plates to determine the cfu associated with the monolayers . Similarly, bacterial adherence to plastic wells was determined in the absence of monolayers . Adherence to the walls was estimated and counts were corrected accordingly .



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Determination of internalised bacteria. Minimum inhibitory concentrations of gentamicin were determined by estimation of growth in overnight broth cultures or on agar plates containing 0 .1-50 µg/ml of gentamicin . Bactericidal concentrations were determined by incubating 10' bacteria in MM in the presence of 10-200 µg/ml gentamicin for periods of 0 .5, 1, 1 .5 and 2 h . Endothelial cells infected as above were washed twice to remove excess non-adherence bacteria and gentamicin was added to a concentration of 125 µg/ml for a duration of 1 h . This treatment eliminated all bacteria in contact with gentamicin . Wells were then further washed with H BSSFBS twice, and lysed in 1% saponin as above for estimation of cfu internalised . In parallel experiments, Huvecs were lysed in distilled water to determine the number of internalised bacteria ; identical results were obtained . Validation of the use of gentamicin . Gentamicin has been the antibiotic of choice for studies on internalisation of bacteria . Although this antibiotic fails to penetrate eukaryotic cells efficient] y, 27 studies on peritoneal macrophages have shown that some uptake does occur in phagocytic cells . 28 In order to minimise intracellular accumulation of gentamicin, both the optimal time of exposure and the optimal concentration of gentamicin for effective extracellular elimination of bacteria were determined . Efficiency of the treatment was confirmed using fixed Huvec monolayers . No bacteria survived gentamicin treatment in the absence of viable host cells . The survivors of gentamicin treatment (125 µg/ml, 1 h) represent true intracellular bacteria since in the presence of the phagocytosis inhibitors, cytochalasin B and cytochalasin D, the numbers that survived the gentamicin treatment decreased by > 95% . Minimum inhibitory concentrations of gentamicin were determined and were found to be identical for b` and b - variants of the same strain and marginally different between strains (2 .5 µg/ml for strain Eagan, 4 µg/ml for strain Rd) . Therefore, in the presence of any intracellular gentamicin, the variants would be expected to be affected similarly . To examine whether gentamicin eliminated all bacteria (that may accumulate) under the monolayer, Huvecs were dissociated from the wells by a brief trypsin treatment, before the addition of gentamicin in MM . Data obtained from such experiments were similar to those obtained by conventional treatment . Results were confirmed by electron microscopy . Association of radio-labelled bacteria . Bacteria were grown on agar plates supplemented with 25 µCi/ml methyl 3 H- thymidine (82 Ci/mmol, Amersham), washed and used for infection of Huvecs either untreated or after treatments described below . At the end of the incubation period, monolayers were washed free of unassociated bacteria and solubilised in 1% SDS in 0 .1 M NaOH . Huvec-associated radioactivity was determined by scintillation counting . Association to plastic wells was also determined and counts were corrected accordingly . Treatment of Huvecs. To determine whether bacteria associated with or entered the endothelial cells in the absence of the host cellular functions, several treatments were carried out prior to analysis as described above . Cold treatment . Both Huvecs and bacteria were cooled to 4°C before addition of bacteria to the monolayers . These incubations were done for a period of 3 h . Bacteria remained viable during this experiment . Fixation . Monolayers were fixed for a period of 30 min in 0 .5% paraformaldehyde in Dulbecco's phosphate buffered saline (PBS), washed three times with PBS prior to infection with bacteria in MM as described . Phagocytosis inhibitors . Cytochalasin B (10 µg/ml) or cytochalasin D (1 µg/ml) was added to Huvecs 30 min prior to infection . Bacteria were added in the media containing inhibitors . Viabilities of bacteria were not affected by cytochalasin treatment . Metabolic inhibitors . Huvecs were incubated with dinitrophenol (1 .5 mm) in 100 µl MM for 30 min, radio-labelled bacteria were added and association was determined (see below) . Inhibition of Huvec function was demonstrated by measurement of 3 H-leucine and 3 H-thymidine uptake . Treatments of bacteria . Treatments were carried out on 3 H-thymidine labelled organisms . Tetracyclin treatment . Bacteria were preincubated for 30 min in the presence of tetracyclin at 2 .5 µg/ml in MM prior to addition to Huvecs in the same medium . This concentration was found to be bacteriostatic both by cfu determination and 3 H-leucine incorporation . Gentamicin treatment was carried out as described above . The antibiotic was present during the subsequent incubation with Huvec monolayers . Dinitrophenol treatment . Bacteria were preincubated with 1

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30 min prior to addition to Huvec in the same medium . Bacterial viabilities declined to below 80% by this treatment compared with untreated controls .

Giemsa staining . Infected monolayers were washed four times and fixed for a minimum of 2 h with 2% paraformaldehyde in PBS and stained with 10% Giemsa stain (BDH) for a period of 1 h . Monolayers were then washed with distilled water and air dried before photography . Electron microscopy. For SEM, Huvecs were seeded on gelatin-coated 13-mm diameter Thermanox coverslips (Flow) in 24-well tissue culture plates . For transmission electron microscopy gelatin-coated polycarbonate filters (0 .4 pm or 3 .0 pm pore size, Transwell, Costar Europe) were additionally used . Coverslips and filters were fixed in 3% glutaraldehyde in 0 .1 M phosphate buffer, pH 7 .3 for a minimum of 3 h . At this stage, the filters were removed from their holders . All samples were post-fixed for 1 h in 2% osmium tetroxide in phosphate buffer followed by dehydration in ethanol . The coverslips for SEM were then critical point dried, mounted on stubs and sputter coated with gold prior to examination with a Philips 505 SEM . The coverslips and filters for TEM were treated with propylene oxide prior to embedding in epoxy resin (EMix) . The monolayers on the coverslips were initially flat embedded and the coverslips removed prior to final embedding . The monolayers from both coverslips and filters were oriented to allow a cross-section through the endothelial cell to be cut . Thin sections were stained with uranyl acetate and lead citrate prior to examination with a Jeol 1000X TEM .

Ruthenium red staining . Filters and coverslips were stained with ruthenium red using the method of Luft ." In this technique, ruthenium red is included in both the primary and postfixatives at a concentration of 0 .1 g% . Because of the dense staining of the glycocalyx of the endothelial cell plasmalemma, this technique made it possible to distinguish between bacteria present in invaginations of the plasmalemma and those located within the cell cytoplasm .

This work was supported by a project . grant from the Wellcome Trust and programme grants from the Meningitis Trust (U .K .) and the Medical Research Council (Grant no . 8325352) . During the period of these investigations, HK was supported by the Wellcome Trust (U .K .), the Royal Society (U .K .), the Sigrid Juselius Foundation (Finland) and the Academy of Finland .

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