Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro

The HIBCPP was maintained as previously described [10, 24]. In brief, HIBCPP were cultured in DMEM/HAM’s F12 nutrient mixture at a ratio of 1:1 (Invitrogen, Carlsbad, Germany) supplemented with 4 mM L-glutamin, 5 μg/ml insulin, 100 U/ml penicillin and 100 μg/ml streptomycin, and additional 15% of heat-inactivated FCS. Cells were seeded on transwell filter inserts (pore diameter 3.0 μm, pore density 2.0 × 10⁶ pores per cm², growth area 0.33 cm²) (Greiner Bio-one, Frickenhausen, Germany) with a density of 0.7 × 10⁵. Hereafter, cells were cultured for two days before turning, and were cultured for up to 5 to 7 additional days before they were used for an experiment.

The measurement of the transepithelial electrical resistance (TEER) and the paracellular permeability provide information about the barrier characteristics. To analyze the barrier function throughout the experiments the TEER was documented by measuring the HIBCPP with a volt-ohm meter using the STX-2 electrode system (Millipore, Schwalbach, Germany) before the infection with bacteria, as well as before and after the TM of leukocytes [16]. Paracellular permeability was determined with dextran-TexasRed (MW 3000, Sigma, Deisenhofen, Germany). Dextran was added to the upper compartment of the filter together with the leukocytes for the 4-h incubation period in a concentration of 100 μg/ml, and flux across the HIBCPP monolayer in the basolateral-to-apical direction was measured. The concentration of dextran in the lower compartment was analyzed with a Tecan Infinite M200 Multiwell reader (Tecan, Männedorf, Switzerland). All measurements were performed in duplicate [16].

The N. meningitidis strain, MC58 WUE 2135 (pil + opc + cap+) [25], and the unencapsulated mutant, MC58ΔsiaD (WUE2425; pil + opc + cap-) [26], as well as the constitutively unencapsulated strain α14 (pil + opc + cap-) [27, 28] were kindly provided by U Vogel and H Claus (Institute for Hygiene and Microbiology, University of Würzburg, Germany), and were cultured as previously described. In brief, aliquots of bacteria were taken from −80°C, and plated on chocolate agar with vitox (Oxoid, Wesel, Germany) and grown at 37°C in 5% CO₂ atmosphere overnight, followed by an additional 90-minute growth period in proteose peptone medium supplemented with 0.042% NaHCO₃, 0.01 M MgCl₂ and 1% PolyViteX (bioMerieux, Lyon, France) until the mid-logarithmic phase was reached and diluted to an optical density (OD)₆₀₀ of approximately 0.1.

Prior to the infection experiments HIBCPP were washed and transferred to medium without antibiotics supplemented with 1% FCS. At this time the cells had a TEER of about 55 Ω × cm² ± 25 Ω × cm². The change of serum concentration leads to a TEER increase up to 200 to 300 Ω × cm² on the following day, and thereafter, cells were used for the TM experiment. HIBCPP on transwell filters were infected from the basolateral cell side (apical filter compartment) with MC58, MC58ΔsiaD or strain α14 at a multiplicity of infection (MOI) of 10. The infection was performed for 2 h at 37°C in 5% CO₂ atmosphere. After the incubation time bacteria were killed by the addition of 100 U/ml penicillin and 100 μg/ml streptomycin to the transwell filters and further incubated for 22 h.

For immune cell transmigration, PMNs and monocytes were isolated from ethylenediaminetetraacetic acid (EDTA)-anticoagulated peripheral blood of healthy adult donors (approval was provided by the local ethics committee of the Medical Faculty of Mannheim, Heidelberg University (2009-327 N-MA)). PMNs were thereafter purified using Polymorphprep™ density sedimentation according to the manufacturer’s instructions (Axis Shield, Liverpool, United Kingdom). The interface containing PMNs was washed with Hank's Balanced Salt Solution (HBSS) with CaCl₂ and MgCl₂ (Invitrogen) followed by an erythrocyte lysis for 15 minutes (lysis buffer contains 8.3% of NH₄Cl (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 1% KHCO₃ (Sigma-Aldrich), 1% (w/v) EDTA (Biochrom AG, Berlin, Germany) dissolved in H₂O and adjusted to 1 l and a pH of 7.2 to 7.4). For TM assays PMNs were loaded with fluorochrome 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetomethyl ester (BCECF-AM; Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions. The purity of isolated PMNs CD66b was 93.99% ± 1.91%.

For monocyte experiments PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation. The negative isolation of monocytes was performed using the Dynabeads® Untouched™ Human Monocytes kit (Invitrogen), according to the manufacturer’s instruction, resulting in a purity of 91.53% ± 1.78% CD14⁺ cells. Monocytes were labeled in a similar fashion as described for PMNs.

To analyze the TM process over HIBCPP, BCECF-AM-loaded leukocytes were added to the upper compartment of transwell filters (blood-side) in a ratio of PMNs:HIBCPP of 10:1 and monocytes:HIBCPP of 1:1, 22 h after previous stimulation of HIBCPP cells with bacteria, TNFα 10 ng/ml, or unstimulated control cells. As a chemoattractant for PMNs, IL-8 (10 ng/ml) (R&D, Wiesbaden, Germany) and for monocytes MCP-1 (50 ng/ml) (R&D) were used and applied in the lower compartment of the transwell filter 30 minutes before starting the TM experiments. After 4 h of TM the HIBCPP transwell filters were transferred for further analysis into a fresh plate, and the fluid within the lower filter compartment was centrifuged (5 minutes, 300 × g) to assure the attachment of PMNs or monocytes to the bottom of the wells. The centrifuged leukocytes were washed twice with HBSS with CaCl₂ and MgCl₂ (5 minutes, 300 × g), lysed with 1% Triton X-100 in PBS and quantified by fluorescence measurement with a Tecan 200 M Infinite Multiwell reader [16]. The remaining supernatant was used in some experiments for cytokine and chemokine, or dextran analysis.

The transwell filters were stained as described previously [16]. In brief, transwell filters were cut out of the insert at the end of the experiment, washed with 1% HIBCPP medium and fixed with 4% formaldehyde for 10 minutes. The filters were permeabilized for 1 h with 0.5% Triton X-100/1% BSA in PBS and incubated with the primary antibodies at 4°C overnight to stain the TJ protein ZO-1 (dilution 1:250) (Invitrogen). Subsequently, filters were stained for 1 h with the secondary antibody (Alexa fluor® 594 goat anti-chicken, dilution 1:250) (Invitrogen), with phalloidin Alexa fluor® 660, dilution 1:60 (Invitrogen) for the actin cytoskeleton staining, with 4'-6-diamidino-2-phenylindole dihydrochloride (DAPI), dilution 1:50000 (Calbiochem, Darmstadt, Germany) and immune cell marker CD14, dilution 1:200 (BD Bioscience, Heidelberg, Germany) for monocytes or CD66b (Lifespan Biosciences, Seattle, WA, USA) for PMNs. After washing with PBS the filters were embedded in ProLong Antifade Reagent (Invitrogen). Subsequently, immunofluoresence analyses were performed using a Zeiss Apotome® with a 63×/1.4 NA objective lens and the Zeiss Scanning Software 4.6 Axiovision software Inside 4D (Carl Zeiss, Jena, Germany).

For the transmission electron microscopic analysis of HIBCPP and leukocytes, filters were fixed after TM for at least 4 h in a 2% glutaraldehyde solution in 75 mM cacodylate buffer (pH 7.4) and washed twice after the incubation time with 75 mM cacodylate buffer (pH 7.4). Subsequently, the support films were removed from the wells using a sharp ophthalmic scalpel. The filters were then cut into strips and post fixed in 1% osmium tetroxide (OsO₄) in cacodylate buffer for 1 h, and dehydrated in ascending series of ethanol and propyleneoxide. For contrast enhancement, they were bloc-stained in uranyl-acetate in 70% ethanol for 4 h and flat-embedded in Araldite (Serva, Heidelberg, Germany). Using an ultramicrotome (Ultracut R, Leica, Bensheim, Germany), semi- (1 μm) and ultra-thin (50 nm) sections were cut. Ultra-thin sections were stained with lead citrate, mounted on copper grids and finally analyzed with a Zeiss EM 10 (Oberkochen, Germany) electron microscope.

For FIB/SEM analysis, the block of the embedded sample was sputter coated with gold palladium and mounted on an appropriate SEM (scanning electron microscope) sample holder. A semi-thin section of the embedded sample was imaged with the light microscope and correlated with the SEM image of the ultramicrotome block face to define the region of interest for three-dimensional analysis. Using a Crossbeam instrument (Zeiss) equipped with a gallium FIB (focused ion beam) and a low voltage SEM, FIB/SEM serial sectioning tomography [29, 30] was accomplished. Thereby, the gallium FIB produces a series of cross-sections containing the region of interest. Each of these cross-sections is imaged by the low keV SEM using the energy-selected backscattered (EsB) electron detector for image acquisition, which yields to images with exceptionally high contrast due to the staining. Additionally, the images show no FIB-induced artifacts, such as curtaining, because of the missing topographical information in backscattered electron images. Using typical voxel sizes in the range of 10 × 10 × 10 nm, morphological information with high spatial resolution is obtained in the volume of interest.

The resulting stack of two-dimensional images was utilized for three-dimensional reconstruction using appropriate software. The open source software ImageJ [31], equipped with the plug-ins StackReg [32] VolumeJ [33] and 3D Viewer [34] was chosen for image processing and three-dimensional reconstruction of the transwell cell culture.

For western blot analysis protein lysates were generated at the end of the TM experiments by extraction of the leukocytes from the upper and lower compartment with modified radioimmunoprecipitation assay (RIPA) buffer (containing 50 mM Tris HCL (pH 8), 150 nM NaCl, 0.1% SDS, 1% Triton X-100, 1% natriumdeoxycholate, 1× protease inhibitor cocktail, 1 mM PMSF, 50 mM NaF, 2 mM EDTA, and 1 mM Na₃VO₄). Moreover, HIBCPP were scraped from filter inserts, centrifuged for 10 minutes at 18000 × g and thereafter lysed as described above. The whole protein content was determined by the Lowry method (DC Protein Assay, BioRad, Hercules, CA, USA) according to the manufacturer’s instruction. The protein samples were mixed with loading buffer and reducing agent (4 to 12%) (Invitrogen). Equal amounts of protein sample were subjected to electrophoresis at 200 V (MOPS running buffer, Invitrogen). The proteins were separated on Bis Tris NuPage® gels (Invitrogen) and afterwards transferred onto a nitrocellulose membrane (0.45 μm, BioRad, USA) by blotting in transfer buffer for 16 h at 20 V and 4°C (Invitrogen). For prevention of non-specific binding of the primary antibody, the membrane was incubated in Tris-buffered saline and Tween 20 (TBST) buffer with 5% milk powder for 1 h. The primary antibodies recognizing β-actin (Sigma, USA) or SIRPα (Abcam, Cambridge, UK) respectively, were incubated overnight at 4°C, and secondary antibodies (anti-mouse and anti-rabbit) were incubated for 1 h at room temperature. The blots were analyzed using Chemi-Smart (Vilbert Lourmat, Torcy, France). For deglycosylation experiments the PNGase F Kit (BioLabs, Ipswich, MA, USA) was used according to the manufacturer’s instructions, using 5 μg or 20 μg of protein depending on the protein lysate sources. Subsequently, loading buffer was added to the deglycosylated proteins and a western blot was performed as described above.

For cytokine/chemokine detection, supernatants were collected immediately after the TM experiments. After centrifugation of the leukocytes, the supernatants were frozen directly at −80°C for later measurement. Samples were analyzed using the Luminex xMAP suspension array technology (Luminex, Austin, TX, USA). A commercially available kit (G-CSF, GM-CSF, GRO (CXCL1,2,3), IL-1α, IL-1β, IL-1ra, IL-6, IL-8, IP-10, monocyte chemotactic protein (MCP)-1, MDC, macrophage inflammatory protein (MIP)-1α, MIP-1β, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, regulated and normal T cell expressed and secreted (RANTES), sCD40L, transforming growth factor (TGF)α, TNFα, vascular epithelial growth factor (VEGF; MPXHCYTO-60 K, Millipore, Germany, for PMN) was used for the detection of different cytokines/chemokines secreted in PMN experiments and another predefined panel of human cytokines/chemokines for the supernatant of monocyte experiments (Eotaxin, Fractalkline, G-CSF, GRO, IL-10, IL-1ra, IL-1α, IL-1β, IL-6, IL-8, IP-10, MIP-1α, MIP-1β, TNF-α; MPXHCYTO-60 K-14 - Human, Millipore, Germany, for monocytes) was used to quantify cytokines according to the manufacturer’s instructions. Samples (25 μl) were used undiluted and were incubated overnight. Standard curves for each cytokine (in duplicate) were generated using the reference cytokine concentrations supplied with this kit. All incubation steps were performed at room temperature in the dark to protect the beads from light. Samples were read on the Luminex 100™ system (Luminex). To avoid between-run imprecision, all samples from the same individual before and after the interventions were measured in the same run. Control samples were included in all runs. The detection limit for any analyte was 3.2 pg/ml with a dynamic range up to 10,000 pg/ml (according to the manufacturer`s instruction). A variety of control samples was included in all runs. The final concentrations were calculated as the average of two independent measures using the IS software version 2.3.

Cell vitality was measured using the Live/Dead® Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, Göttingen, Germany) according to the manufacturer’s instructions. The results were photo-documented by fluorescence microscopy. Cytotoxicity was not observed in any of the experiments (data not shown).

All statistical calculations have been performed using the SAS system, release 9.2 (SAS Institute Inc., Cary, NC, USA). Quantitative parameters are presented as mean values and SD. For approximately normally distributed data (for resistance or TM rate), one-way analysis of variance (ANOVA) was performed to compare the mean values between several groups. In the case of a significant test result, pairwise comparisons were made using Scheffé’s test, or (to compare to a single control) Dunnett’s test. For these analyses, the SAS procedure PROC MIXED was used with the experiment and amount of filters used as random factors. For skewed data (on permeability), the Kruskal-Wallis test was used followed by the Mann–Whitney U-test for pairwise comparisons. A test result was considered statistically significant for P-values below 0.05.

First we analyzed the effect of PMN on the HIBCPP barrier function with or without bacterial stimulation. We observed an overall increase of TEER after 24 h of stimulation under all conditions (Figure 1A). Thereafter, PMNs were applied in the upper compartment of the culture insert with or without additional IL-8 in the lower filter compartment. We observed a significant increase in PMN TM in the presence of IL-8 (3.01% ± 0.45%) compared to the TM without IL-8 (1.5% ± 0.37%) (data not shown).

Next we analyzed barrier function and TM of PMNs after infection of HIBCPP with N. meningitidis. After the 4-h PMN TM period we observed a significant TEER decrease under all conditions (Figure 1A). The paracellular permeability remained low under all conditions, but was significantly lower under control conditions compared to stimulated cells (Figure 1B). Importantly, we demonstrated a significant increase in TM after stimulation with strain MC58 and TNFα, whereas the TM remained in the range of control values after stimulation with strain MC58ΔsiaD and strain α14 (Figure 1C). In Live/Dead assays no significant alteration of HIBCPP or PMN viability was observed (data not shown).

In the next step we analyzed monocyte transmigration across HIBCPP and its effect on the barrier function after infection with N. meningitidis. First we investigated monocyte TM in the presence or absence of the chemoattractant MCP-1 (0.046% ± 0.075%) and demonstrated a significant increase in monocyte TM in the presence of MCP-1 (1.02% ± 0.031%) (data not shown). After bacterial infection of HIBCPP and monocyte TM we observed no significant change in barrier function (Figure 2A, B). In contrast to the PMN TM, the migration of monocytes was significantly inhibited by bacterial infection, whereas TNFα had no influence compared to unstimulated controls (Figure 2C). No significant alteration of HIBCPP or monocyte viability was observed (data not shown).

During bacterial meningitis PMNs display the first line of immune defense followed by monocyte infiltration as the second wave of inflammatory response [35]. Consequently, we studied whether the presence of PMNs can influence the TM of monocytes over the plexus epithelium after bacterial infection with N. meningitidis. Additionally, as known from previous work, PMN granule proteins can promote monocyte recruitment and work as chemoattractants inducing also the secretion of MCP-1 or IL-8 [36]. Therefore we performed co-culture PMN/monocyte TM experiments. PMNs were applied for 2 h into the apical transwell filter compartment; afterwards monocytes were added for another 2 h and the final TM rate of monocytes was analyzed. In these co-culture experiments monocyte TM after bacterial stimulation with MC58 was significantly increased in the presence of PMN compared to monocyte TM alone (Figure 3).

In the following experiments we analyzed the TM pathway used by the PMNs and monocytes to overcome the plexus epithelium. Therefore, we performed extensive immunofluorescence, and electron microscopic studies, as well as FIB/SEM studies to elucidate the TM process in more detail. Three-dimensional immunofluorescence analysis revealed that PMNs migrate via the para- (Figure 4) and transcellular (Figure 5) route through HIBCPP. The first immunofluorescence series shows paracellular migrating PMNs, which squeeze through the TJs (Figure 4A-E). In Figure 4A the PMN is about to exit the intercellular space, where the ZO-1 strands are interrupted; in Figure 4B and C the PMN has almost left the HIBCPP cell through a widened intercellular space. In the latter image the intercellular gap formation was observed almost from the top to the lower level of the HIBCPP cell. In Figure 4D the PMN is squeezing between TJs at a multicellular corner and in Figure 4E the PMN is covered by ZO-1 from two sides in parallel, indicating direct traversal through the tight junctions.

In the case of transcellular TM, PMNs migrated in a clear distance to the cell borders (Figure 5). Here, no TJ alterations were found in different stages of PMN traversal through the epithelial cell from the basolateral to the apical side. As seen in Figure 5A and D, the migrating PMNs are found above the cell level with only a part within the cell. The side views illustrate that the PMN is about to leave the cell. In Figure 5C the major part of the PMN is found already outside of the cell and the PMN is surrounded by TJ strands, whereas in Figure 5B and E the PMN is still located within the HIBCPP cell body.

In the next step the actin morphology during this TM process of PMN and monocytes was further analyzed, showing no differences between stimulated HIBCPP cells and control conditions (Figure 6). Moreover no major differences of actin morphology in PMN and monocyte experiments were observed. Interestingly, HIBCPP showed a tubular morphology after bacterial stimulation, in contrast to TNFα-stimulated and control cells, which had a rather flat character.

Electron microscope investigations were performed to provide further evidence for the mode of TM of PMN through the HIBCPP monolayer. We compared ortho-graded filters that were sectioned almost parallel through the apical surface, to allow better interpretation of cellular details during TM. Here, as in the immunofluorescence analyses, we showed that PMNs can migrate either via the paracellular route (Figure 7A, B) or via the transcellular route through the epithelial cell itself (Figure 7C, D). In Figure 7A and B the PMN is squeezing through the paracellular space of two epithelial cells indicated by open hemidesmosomes, which are marked with arrows on each side. Figure 7C and D show intact TJs, which are marked with an arrow underlying the clear distance to the PMN.

With the help of the FIB/SEM Crossbeam analysis, paracellular-migrating PMN (Figure 8A Additional file 1: video 1) and transcellular-migrating PMN (Figure 8B Additional file 2: video 2) were also identified. This technique provides the possibility of three-dimensional electron microscopic analysis of the whole cell monolayer. Here we show a PMN, which migrates via the paracellular route between the epithelial plexus cells (Figure 8A) through the TJs (arrows indicate the cell borders), as well as in a clear distance to the cell borders (arrows) directly in the cytoplasm (Figure 8B).

Due to the low TM rate of monocytes, which was significantly reduced after bacterial infection, the usefulness of immunofluorescence and especially, electron microscopic analysis to clarify the TM route of monocytes was limited. Therefore, we focused on immunofluorescence studies of TNFα-stimulated and unstimulated control HIBCPP (Figure 9). In control and TNFα-stimulated cells we observed paracellular-migrating monocytes, which were frequently found to migrate at bi- or multicellular corners (Figure 9A, B) as well as transcellular ones (Figure 9C, D). In Figure 9A the monocyte is exiting the cell via the paracellular route, whereas Figure 9B displays a monocyte entering the paracellular migration route. Furthermore transcellular transmigrating monocytes were found in the HIBCPP cell body and at a clear distance from the cell borders (Figure 9C, D).

To analyze whether a distinct cytokine/chemokine pattern is responsible for the differential TM pattern of PMNs and monocytes a cytometric bead array was performed with supernatants of PMN and monocyte experiments as described above. The first group of cytokines/chemokines including IL-6, MIP-1α, MIP-1β and IL-1β showed significantly increased secretion levels after the TM of PMNs through infected HIBCPP (Figure 10A-D), whereas another group of cytokines such as GRO, MCP-1, IP-10 and RANTES (Figure 10E-H) was shown to be released by HIBCPP already after stimulation with different N. meningitidis stains alone. TNFα was used again as the positive inflammatory control. TNFα (Figure 10I) and IL-8 (Figure 10J) were significantly produced after bacterial stimulation of HIBCPP, which was further enhanced after the PMN TM.

As in the PMN analyses the detected cytokines/chemokines in monocyte TM experiments were classified in different categories of their secretion pattern after the different stimuli. IL10, IL1α, IL1β, IL-6 and TNFα (Figure 11A-E) were mainly released after monocyte TM in combination with bacterial stimulation, whereas GRO and IL-8 (Figure 11F-G) were already secreted at higher levels only after bacterial stimulation of HIBCPP alone. Another group displayed chemokines, such as, MIP-1α and MIP-1β (Figure 11H-I), which were strongly secreted by monocytes alone after bacterial stimulation. This secretion was further enhanced by the presence of HIBCPP.

Comparing the cytokine bead array (CBA) results of the monocyte and PMN TM experiments, we observed, that the cytokine/chemokine levels secreted during PMN migration differed significantly from those observed during monocyte migration. Chemokines, such as, MIP-1α and MIP-1β (Figure 10B,C and Figure 11H, I), as well as cytokines, such as IL-1β (Figure 10D; Figure 11C), are secreted in concentrations that were up to 10 times higher during monocyte TM in combination with bacterial stimulation, than during PMN TM. Moreover, MIP-1α, MIP-1β (Figure 10B,C and Figure 11H, I) and IL-1β (Figure 10D and Figure 11C) were also released at levels around 10 times higher after bacterial stimulation of monocytes without HIBCPP compared to PMN. Both GRO and IL-8 had similar secretion levels in both settings (Figure 10E, J and Figure 11F, G).

In addition to the cytokine/chemokine analysis we wanted to determine possible binding partners of PMNs and monocytes involved in the TM process. As already known from previous studies, SIRPα was shown to mediate migration of leukocytes through endothelial [37, 38] and epithelial cells [39]. Therefore we aimed to investigate the possible involvement of SIRPα in the differential TM of PMNs and monocytes by western blot analysis. We determined the deglycosylation of SIRPα after bacterial infection, after TNFα stimulation, and under control conditions. As shown in Figure 12 the neuronal form of SIRPα is expressed at about 85 kDa under all conditions. Interestingly, SIRPα was deglycosylated in monocytes after bacterial infection, whereas under TNFα stimulation and under control conditions only a very slight band of the deglycosylated form was observed. In contrast, only the neuronal form of SIRPα was observed in PMN experiments, as well as in experiments with HIBCPP only (data not shown).