Skip to main content
NCBI home page
Journal of Anatomy logoLink to Journal of Anatomy
. 2010 Sep 29;217(6):740–754. doi: 10.1111/j.1469-7580.2010.01304.x

Macrophages increase microparticle uptake by enterocyte-like Caco-2 cell monolayers

Siobhan M Moyes 1, John F Morris 1, Katharine E Carr 1
PMCID: PMC3039186  PMID: 20880316

Abstract

Caco-2 cells form an enterocyte-like monolayer that has been used to explore small intestinal microparticle uptake. They are a useful functional model for the investigation of in vivo drug delivery systems and the uptake of particulate environmental pollutants. The aim of this paper was to determine if the previously reported decrease in Caco-2 transepithelial resistance following exposure to macrophages was matched by increased microparticle uptake, especially as macrophage phagocytosis simulates removal of particles from the subepithelial compartment. Caco-2 cells were grown as a monoculture for 21 days on insert membranes. A compartmentalised model involved Caco-2 cells in the upper compartment, with THP-1-derived macrophages adhering to the base of the underlying well, the two cell populations communicating only through the shared culture medium. Caco-2 cells were also cultured in macrophage-conditioned medium and all groups were exposed apically to 2 μm latex particles for 5 or 60 min. Parameters measured were: transepithelial resistance; cytokine levels; cell dimensions and the distribution of nuclei, actin and junctional proteins. Subepithelial particle numbers, defined as those located below the insert membrane, were also counted and were significantly increased in the Caco-2/macrophage model, with over 90% associated with the macrophages. Other changes induced by the presence of macrophages included decreased transepithelial resistance levels, diffuse localisation of some junctional proteins, higher proinflammatory cytokine levels, disorganisation of cell shape and decreased cell height associated with actin reorganisation. Macrophage-conditioned medium produced a smaller transepithelial resistance decrease than the Caco-2/macrophage model and there were few other changes. In conclusion, culture of Caco-2 cells with underlying macrophages produced a lower, less organised epithelium and greater microparticle uptake.

Keywords: Caco-2 cells, cell dimensions, cytokines, immunofluorescence, microparticle uptake, THP-1-derived macrophages, transepithelial resistance

Introduction

The Caco-2 cell monoculture is a valuable model of small intestinal epithelium because, despite its colonic adenocarcinomatous origin, the cells behave like enterocytes (Delie & Rubas, 1997), which are the principal epithelial cells on the villous surface. The model is useful for the investigation of microparticle uptake through the intestinal epithelium. This process in vivo has been regarded for some time as ‘neither exceptional nor unusual’ with respect to encapsulated drug delivery (Florence, 1997) and as useful for predicting possible mechanisms for uptake of microparticles and also of the increasingly relevant nanoparticles (Powell et al. 2010).

Microparticle uptake is also important for environmental toxicology (Hodgson et al. 2005; Stather, 2007) including exposure to radioactive particles such as those produced by the Chernobyl disaster in 1986, when the smaller particles were 0.3–1.5 μm in diameter and the larger 10 μm (Cuddihy et al. 1989). Inert latex particles in the range 2–10 μm were taken up through the intestinal wall of rats in an in vivo in situ model (Hodges et al. 1995; Carr et al. 1996). The 2 μm latex microparticles are themselves much larger than digested dietary subunits, which are so small that their dimensions are indicated by their molecular weights and therefore given as kilodaltons. The microparticles on the other hand are more of a size with subcellular cytoplasmic structures such as mitochondria and are much smaller than the individual enterocytes, which apically measure 33 × 17 μm for Caco-2 cells in vitro (Moyes et al. 2007).

Quantitative assessment of microparticle uptake into small intestinal tissues for laboratory animals varies with technique but in general ranges from 0.1 to 4.3% after maceration and from 0.04 to 0.32% after microscopy (Hodges et al. 1995; Doyle-McCullough et al. 2007): these figures are similar to those from other groups, who reported 0.01% for smaller particles (Ebel, 1990) or 3.6% (Jani et al. 1990).

The sightings of microparticles in the intercellular position with in vivo in situ models (Smyth et al. 2005) made it important to explore whether a possible paracellular uptake route across the epithelium could involve the ‘unzipping’ of villous tight junctions (TJs) in a manner similar to that reported for the interaction between Caco-2 cells and antigen-presenting cells (Rescigno et al. 2001). The Caco-2 in vitro model allows exploration of this question as microparticles travel through to the lower well below the insert to a similar extent to the in vivo in situ model (Moyes et al. 2007). In addition, in vitro epithelial permeability was assessed by measuring transepithelial resistance (TER). The 2 μm latex particles could also be considered an epithelial permeability marker and the 3 μm pores of the insert membranes allow their passage through to the lower well where they can be counted. It has also been confirmed that such pores, unlike those of larger sizes, do not increase the chances of multilayering of the epithelium above and below the membrane (Delie & Rubas, 1997; Moyes et al. 2007).

Caco-2 cell TJs become more permeable after exposure to external irradiation or ethanol and, at similar time points after microparticle addition, the number of particles passing through also increases (Moyes et al. 2008, 2010). The Caco-2 TJs are also known to open if there were macrophages in a separate underlying compartment (Kanzato et al. 2001). This forms a ‘compartmentalised’ model rather than a co-culture situation, as the two populations are separated by the insert membrane and are in communication only through shared medium fluid. Macrophages are particularly appropriate for compartmentalised culture and particle uptake studies because they contribute to a possible onward route for particle removal in vivo (Wells et al. 1988). They are also used in phagocytic assessment of implant debris, where the outcome depends on particle load, aspect ratio and chemical reactivity (Hallab & Jacobs, 2009): particles must also be smaller than 10 μm to produce an inflammatory response, a criterion met by the current protocol. The phagocytic nature of macrophages thus improves the usefulness of the Caco-2/macrophage model for in vitro study of microparticle uptake.

The aim of the current paper was to determine whether the macrophage-induced TJ opening between intestinal epithelial cells in culture is accompanied by alterations in other aspects of the epithelial monolayer and in the extent of latex microparticle uptake across it.

Materials and methods

Protocol outline

Caco-2 intestinal epithelial cells were cultured on a microporous membrane either alone as a monoculture or exposed to macrophages for 1–72 h (‘Caco-2/macrophage group’). For both groups, and macrophage monocultures, cytokine levels were measured and structural features recorded using immunofluorescence and confocal microscopy. The Caco-2 monoculture was also exposed to macrophage-conditioned or untreated culture medium for 24 h (‘macrophage-conditioned medium’ and ‘untreated medium’ groups).

Caco-2 cells in all groups were then exposed apically via the upper well medium to 1 μL (5.68 × 106particles μL−1, Polysciences Inc.) of 2 μm diameter (SD ± 0.089 μm) Fluoresbrite® yellow-green latex particles for 5 or 60 min (Table 1, Fig. 1). The Caco-2 monoculture was thereafter designated the ‘particle-only’ group, and the term ‘combined’ was used for Caco-2 cells exposed to both pre-treatment and thereafter to microparticles. The effect of these treatments was measured using transepithelial resistance and particle uptake assays. At the end of the experimental procedure, cell morphology was compared for Caco-2 cells across groups. Particles associated with macrophages were also quantified by both UV and confocal microscopy.

Table 1.

Treatment groups and assays.

Treatments/assays
Treatment groups Assays Treatment Assays Treatment Assays
Particle-only groups Control/TER measured on day 21 Some samples prepared for immunofluorescent labelling and cytokine measurement – – 5 or 60 min particle exposure Post-particle/TER Some samples prepared for immunofluorescent labelling Sub-membranous particle counts
Caco-2/macrophage compartmentalised culture groups Pre-treatment/TER measured before Caco-2/macrophage compartmentalised culture (days 18–21) Caco-2/macrophage compartmentalised culture for 1–72 h Post-treatment/TER Some samples prepared for immunofluorescent labelling and cytokine measurement 5 or 60 min particle exposure Post-particle/TER Some samples prepared for immunofluorescent labelling Sub-membranous particle counts
Medium transfer groups Pre-treatment/TER measured before medium transfer on day 20 24 h exposure to macrophage-conditioned medium or untreated-medium Post-treatment/TER Some samples prepared for cytokine measurement 5 or 60 min particle exposure Post-particle/TER Sub-membranous particle counts
Open in a new tab

The treatment group names are given in column 1. The particle-only groups were stored in the incubator until their control/TERs were measured (where TER is transepithelial resistance), after which particles were immediately added.

Fig. 1.

Fig. 1

Open in a new tab

Schematics of the Caco-2/macrophage compartmentalised culture model. The solid arrows above the timeline from 72 to 1 h indicate the beginning of the Caco-2/macrophage compartmentalised culture period, which was preceded by a pre-treatment/TER measurement for all groups, where TER is transepithelial resistance. The control/TER value was measured on particle-only samples on day 21 of Caco-2 culture immediately before particle addition (dashed arrow). The diagram of the cell culture model shows Caco-2 cells cultured on Transwell® inserts with 3 μm pores, with THP-1-derived macrophages adhering to a glass coverslip at the bottom of the six-well receiver plate. The two cell populations communicated only through the shared culture medium.

Cell culture protocols

Caco-2 cell culture

ATCC Caco-2 cells (HTB-37™, passage 28–39), were cultured in a 37 °C incubator with a 5% CO2 atmosphere in ATCC-formulated Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS) Gold (PAA Laboratories) and 1% penicillin/streptomycin (Gibco®). The cells were seeded at a density of 1 × 106 cells per six-well polyester Transwell® insert (3 μm pores, Corning Costar) and were cultured to confluence over 21 days, although some groups were exposed to treatments during days 18–21 (Fig. 1). Cell culture medium in the upper and lower wells (1.5 and 2.5 mL, respectively, Fig. 1) was replaced every 2–3 days and on day 20 of culture.

THP-1 culture – preparation of macrophages from monocytes

The human, monocytic, THP-1 cell line (ATCC, TIB-202™) was cultured in suspension in ATCC-formulated RPMI-1640 complete growth medium supplemented with 10% FBS gold, 0.05 mm 2-mercaptoethanol (Sigma) and 1% penicillin/streptomycin. These cells were transformed to an adherent macrophage phenotype by resuspension in THP-1 medium, supplemented with 100 nm phorbol 12-myristate 13-acetate (PMA; Sigma: Inoue et al. 1999), seeded at a density of 1 × 106 cells per well (six-well format) containing a sterile 24-mm diameter glass coverslip, previously serum coated for a minimum of 2 h. These cells were then cultured at 37 °C for 24 h, during which time the THP-1 cells adhered to the surface of the coverslips and wells and were termed ‘macrophages’.

Experimental groups

Control/TER values and morphology-control group

‘Control/TER’ values referred to the measurements taken on day 21 for the untreated group before the addition of particles (particle-only group). The term ‘morphology-control’ refers to the reference group for cell dimensions, subjected to transepithelial resistance but not exposed to particles (Moyes et al. 2010).

Particle-only group

Caco-2 cells were exposed only to particles for 5 or 60 min and were designated particle/only-5 and particle/only-60, respectively.

Caco-2/macrophage compartmentalised culture group

Individual Transwell® inserts containing Caco-2 cell layers were transferred into six-well receiver plates pre-seeded with THP-1-derived macrophages, which by then had adhered to the base of the well (Fig. 1, Section THP-1 culture – preparation of macrophages from monocytes). Both cell populations were then cultured in fresh Caco-2 medium for 1–72 h at 37 °C until day 21 of Caco-2 culture. Cells also received a medium change on day 20 (Fig. 1). The data sets examined were those for the 24-h groups, which were subsequently exposed to particles for 5 or 60 min (‘macrophage/particle-5’ or ‘macrophage/particle-60’, respectively).

Medium transfer groups

To produce ‘macrophage-conditioned medium’, THP-1 monocytes were seeded at a concentration of 2.2 × 105 cells per cm2 in a 75-cm2 flask and transformed to macrophages, as above. The THP-1 medium was then discarded and replaced with 32 mL of Caco-2 culture medium for 24 h. This medium was then removed from the cells and filter-sterilised immediately before addition to the Caco-2 model. The ‘untreated-medium’ consisted of filter-sterilised Caco-2 medium warmed to 37 °C to bring it to the same temperature as the cells. Caco-2 cells were incubated at 37 °C for 24 h with ‘macrophage-conditioned medium’ or ‘untreated medium’ (Table 1), before particle addition. This produced groups designated as ‘macrophage-conditioned medium/particle-5’, ‘macrophage-conditioned medium/particle-60’, ‘untreated medium/particle-5’ and ‘untreated medium/particle-60’.

Assays, sample preparation and data handling

Transepithelial resistance data collection

Before cell seeding, the transepithelial resistance (TER) of the insert membrane with culture medium was recorded (TERInsert) using a Millicell®-ERS instrument (Millipore). At the beginning of each experiment, the transepithelial resistance for the Caco-2 epithelium on the insert was measured: for quality control purposes data were only used if the control/TER raw measurement was at least 70 Ω higher than TERInsert (Moyes et al. 2007). Transepithelial resistance measurements were calculated in Ohms cm2 (Ω·cm2) to determine the transepithelial resistance of the epithelium alone (Wells et al. 1998) and allow for its total surface area, using the following equation; [TER–TERInsert] × 4.5 cm2. The Millicell-ERS meter and electrodes were tested intermittently throughout each experiment for deviation from the manufacturer's control parameters: no drift was observed. Laminar flow hood temperature was also recorded. Data measurements were taken from two to five independent experiments.

Calculation of delta changes in transepithelial resistance

Delta changes in transepithelial resistance were obtained by subtracting a transepithelial resistance from an earlier or reference value, such as the control/TER. An increased transepithelial resistance was taken to indicate tighter TJs and a decreased transepithelial resistance looser TJs. The effect of particle exposure alone was calculated as a delta change by subtracting the post-particle/TER measurement from the control/TER. The effect of compartmentalised culture with macrophages or medium transfer was calculated by subtracting either pre-treatment/TER from post-treatment/TER, or control/TER from post-treatment/TER, as appropriate. The effect of subsequent particle addition was calculated either (i) for the ‘combined effect’, by subtracting the post-particle/TER from the control/TER, or (ii) for the ‘particle effect’, separating particle and treatment effects, by subtracting the post-particle/TER from the post-treatment/TER.

Other methods of recording changes in transepithelial resistance

Transepithelial resistance changes could also be calculated as percentages of the relevant reference value. Delta changes were used for numerical comparisons across the treatment groups described in this paper. Table 2 contains raw transepithelial resistance values.

Table 2.

Transepithelial resistance data before and after particle addition.

Transepithelial resistance (Ω · cm2)
Treatment group Raw data for Control/TER Raw data for post-treatment/TER Delta changes after pre-treatment Raw data for post-particle/TER Delta changes after particle exposure
Particle-only groups
 Particle/only-5 451 ± 19 – – 580 ± 25 134 ± 7#
 Particle/only-60 451 ± 19 – – 484 ± 31 27 ± 12P/O-5
24 h Caco-2/macrophage groups
 Macrophage/particle-5 Control/TER subset 144 ± 12 −438 ± 31# 213 ± 6 77 ± 6†
 Macrophage/particle-60 Control/TER subset 144 ± 12 −438 ± 31# 206 ± 15 56 ± 4
24 h macrophage-conditioned medium groups
 Macrophage-conditioned medium/particle-5 Control/TER subset 314 ± 31 −221 ± 41#C/M 450 ± 41 125 ± 11P/O-5,M/P-5
 Macrophage-conditioned medium/particle-60 Control/TER subset 314 ± 31 −221 ± 41#C/M 346 ± 53 43 ± 11
24 h untreated-medium groups
 Untreated-medium/particle-5 Control/TER subset 566 ± 10 31 ± 10M-CM 736 ± 15 174 ± 12†P/O-5,M-CM/P-5
 Untreated-medium/particle-60 Control/TER subset 566 ± 10 31 ± 10M-CM 639 ± 20 68 ± 10†
Open in a new tab

Mean transepithelial resistance (TER) values ± standard error of the mean (SEM) for n ≥ 21 samples are calculated from a minimum of two experiments. Pre-treatment delta changes (italics) in TER following treatments are calculated well by well as [pre-treatment/TER − mean control/TER from the same experiment]. Particle associated delta changes (italics) in TER between treatment and particle exposure are calculated well by well as [post-particle/TER − post-treatment/TER]. Statistical differences (P ≤ 0.05) for delta changes are illustrated as ‘#’ a difference from control/TER, ‘†’ from post-treatment/TER, ‘C/M’ from the Caco-2/macrophage effect, ‘P/O-5’, ‘M/P-5’ and ‘M-CM/P-5’ from particle/only-5, macrophage/particle-5 and macrophage-conditioned medium/particle-5 respectively.

Cytokine quantification

The culture medium from Caco-2 and macrophage monoculture and also from the 24 h Caco-2/macrophage model was assayed for the following cytokines; interleukin (IL)-1β, -6, -8, -9, -10, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. The fluid taken from macrophage monocultures for cytokine measurements was in fact the same as that described elsewhere in this paper as macrophage-conditioned medium. Cytokine quantification was carried out at the University of Sheffield's Flow Cytometry Core Facility, using the Becton Dickinson Cytometric Bead Array™ Human Soluble Protein Flex Set Assay procedure according to manufacturer's guidelines. The sample data were acquired on a BD FACSArray™, with BD FACSArray™ software and analysed using FCAPArray™ software. The standard curve detection range was between 0 and 2500 pg mL−1 for all cytokines assayed.

Immunofluorescence experiments for junctional proteins and actin

These were carried out on Caco-2 cells cultured alone and on the 24 h Caco-2/macrophage model, with or without particle exposure for 60 min. Caco-2 cells cultured on insert membranes were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and antigen retrieval was carried out with sodium citrate buffer (Shi et al. 1993). Cells were then permeabilised in 0.1% Triton X-100 for 15 min and blocked for 30 min in 3% bovine serum albumin. Samples were incubated with the primary antibodies rabbit anti-occludin (1 ng μL−1; Invitrogen 71–1500), rabbit anti-ZO-1 (25 ng μL−1; Invitrogen 61–7300) or mouse anti-E-cadherin (500 ng μL−1; Abcam ab1416), for 2 h. After washing, the cells were incubated for 1 h with biotinylated goat anti-rabbit IgG or biotinylated horse anti-mouse IgG (15 ng μL−1; Vector Laboratories) and then exposed to a mixture of fluorescein Avidin-D (20 ng μL−1; Vector Laboratories) and TRITC-labelled phalloidin-tetramethylrhodamine B for F-actin (20 ng μL−1; Sigma 77418), in PBS for 45 min. They were then washed and mounted using Mowiol mounting medium containing DAPI. Negative controls were also carried out omitting either the primary antibody or the actin stain.

All images were taken using a Zeiss LSM510 or 710 confocal microscope and analysed with either LSM Image Browser or ZEN 2009 LIGHT EDITION software, respectively, for the distribution of the junctional proteins and cytoplasmic F-actin in order to compare normal and tall epithelium with pleomorphic or shorter regions. For junctional protein distribution, staining was analysed in a minimum of six sites of three samples from two to three experiments. Low- and high-power z-stack images (×20, or ×63 with ×2 digital zoom) were taken to record the uniformity of the staining and more detail, respectively. For cytoplasmic actin distribution, between 10 and 25 cells were analysed from three samples. Macrophages cultured on coverslips in the well below the Caco-2 cells were also fixed in 4% paraformaldehyde, labelled for F-actin and screened on the confocal microscope as above. Analyses of the height of 8–10 cells over three experiments were carried out using confocal z-stacks, and the relative locations of the junctional proteins, actin, nuclei and latex particles were recorded.

Cell dimensions

For particle-only, Caco-2/macrophage and macrophage-conditioned medium groups, 5 and 60 min after particle exposure (n = 27), confocal microscopy was used to measure cell height and widths, both major and minor, of apical and basal aspects (Moyes et al. 2008, 2010). Data for the particle-only group were also compared to those for the previously published morphology-control group, which was exposed only to two transepithelial resistance measurements 75 min apart (Moyes et al. 2010).

Microparticle analysis

The effect on microparticle aggregation was studied after suspension in either distilled water or Caco-2 culture medium. Latex particles (1 μL with 5.65 × 106) were added to 1.5 mL of water or medium and 0.5 mL placed on a slide for examination by UV microscopy. The numbers of single or aggregated particles were counted in four sites from three samples and the percentage of aggregates calculated (n = 12).

‘Sub-membranous’ particles, for particle-only, Caco-2/macrophage and macrophage-conditioned medium groups, were defined as all those located below the insert membrane at the end of the experiment (Fig. 1). These included those washed off the basal aspect of the insert membrane, those in the lower well medium as previously described (Moyes et al. 2010) and also, for the compartmentalised model, those associated with macrophages. The macrophage-covered glass coverslips were fixed with 3% glutaraldehyde in PBS for at least 20 min and mounted on coded microscope slides using Vectashield with propidium iodide (Vectorlabs). Particles were counted using a previously defined crosswire method (Moyes et al. 2007) and the numbers multiplied by 15.9 to account for the entire surface area.

Data analysis

Following the Levene's test showing homogeneity of variance, group anova and Student's t-test statistical analyses were carried out on the transepithelial resistance, particle numbers and cytokine data, as measured or logarithmically transformed. For the cell dimension data, as the Levene's test did not show homogeneity of variance, the non-parametric Kruskal–Wallis anova and Mann–Whitney U-tests were carried out. Quantitative results were included if they were significantly different (P ≤ 0.05) with respect to the values of reference groups.

Results

Monoculture controls

The quality control procedures based on the control/TER values led to fewer than 1% of the samples being discarded. The hood temperature varied from 21 to 24 °C, with a mean value of 22.8 °C. Three experiments in which a Caco-2/macrophage culture time course was carried out had a mean pre-treatment/TER value increasing from 477 ± 25 to 623 ± 17 Ω cm2 during days 18–20 (P = 0.0001) and 555 ± 14 Ω cm2 on day 21 (n = 24 from three independent experiments). As a result, for the time course experiments all post-treatment/TER values were compared to the mean control/TER on day 21. When the mean control/TER for these three experiments were pooled with a further two experiments focusing on the 24 h Caco-2/macrophage time point (n = 24) the mean control/TER for day 21 was reduced to 451 ± 19 Ω cm2 (n = 48, Table 2).

Immunofluorescence labelling and orthogonal views from confocal microscopy z-stacks of Caco-2 cells confirmed the columnar nature of the cells (Fig. 2). No staining was visible in any of the negative controls. The tight junctions were identified by the labelling for occludin and its associated ZO-1, which outlined the polygonal nature of the cells and was confined to a discrete apical belt position on the lateral aspect of the cells just below the microvilli, identifiable by their actin cores. At the site of some junctions between several cells there were bright, spherical accumulations of labelling, representing aggregations of junctional protein (Fig. 2). Most of the cells were apically regular in shape and of a standard size, although there were a few areas where this was less obvious: each cell was surrounded on average by six cells. The predominance of regular cell packing was continued basally and visualised through the presence of the adhering junction protein E-cadherin, which was linear apically but increasingly punctate more basally along the lateral wall.

Fig. 2.

Fig. 2

Open in a new tab

Immunofluorescence of untreated Caco-2 cells. These cells were labelled with occludin (left), ZO-1 (middle) or E-cadherin (right) junctional proteins (FITC-green staining), actin (TRITC-red staining) and a DAPI nuclear counter-stain (blue). Top row illustrates a merged image of all three fluorescent labels taken with a ×25 oil immersion objective lens on a Zeiss LSM510 confocal microscope. Second row is the same image with only the junctional protein displayed to illustrate the uniform staining at low power. Third row, merged images at a higher magnification (×63 oil) for correlation with fourth row orthogonal view of the same area. Darker areas are not indicative of epithelial discontinuities but represent either variations in height of the cells or the supranuclear actin-free region. In the orthogonal view the arrow marks the level of the picture just above it: membrane pores are illustrated to varying degrees, although the z-stacks were always continued down until they were visible. These 12 images collectively display the regular arrangement of cells, basal nuclei, location of actin and position of junctional proteins. The bright, spherical aggregations of junctional protein are best illustrated in the second image of the third row.

Actin staining (Fig. 2) was observed as a closely packed layer, parallel to the apical surface and approximately 3.2 μm thick. This began at or near the level of the tight junction proteins and then extended below them. Below this, the staining became more diffuse, leaving substantial cytoplasmic regions, apical to the nuclei, entirely without actin. Most nuclei were arranged regularly and basally, together with a co-localising actin region approximately 6.5 μm deep. A layer of actin was also located along the entire lateral walls, at the membrane or at its cytoplasmic aspect. The double layer of membrane and actin in two adjacent cells was approximately 2 μm thick. In tall, well organised epithelium these layers were usually straight and at right angles to the apical surface but were sometimes slightly oblique and/or occasionally irregular.

Only four of the cytokine assays gave positive results (Fig. 3) and IL-9, IL-10 and IFN-γ were not identified in the medium of any of the treatment groups. Macrophages alone produced significantly more IL-1β and IL-8 and less IL-6 compare with Caco-2 cells.

Fig. 3.

Fig. 3

Open in a new tab

Cytokine production by different groups of cells. The concentrations of IL-1β, IL-6, IL-8 and TNF-α in pg mL−1 were analysed by the BD CBA® Human Soluble Protein Flex Set Assay according to manufacturer's guidelines for Caco-2 or macrophage cells cultured alone or for Caco-2/macrophage compartmentalised culture. The fluid taken from macrophage monocultures for cytokine measurements is in fact the same as that described elsewhere in this paper as macrophage-conditioned medium. Significant differences (P ≤ 0.05) with P-values are illustrated by solid lines between treatment groups. For most cytokines measured, the dual culture levels differ from those of both monocultures.

Exposure of Caco-2 cells to macrophages

Regardless of whether the reference value was control/TER or pre-treatment/TER (data not shown), all Caco-2/macrophage compartmentalised culture times, apart from that at 1 h, showed a marked transepithelial resistance decrease (P = 0.0001, Fig. 4). The maximum decrease for the Caco-2/macrophage model occurred after 12 or 24 h (P = 0.0001): the latter was selected as the principal treatment time for use in subsequent experiments. The transepithelial resistance values for the macrophage-conditioned and untreated medium groups were also little changed by the choice of reference group or calculation (data not shown). Exposure to macrophage-conditioned medium for 24 h decreased the Caco-2 cell transepithelial resistance less than compartmentalised culture (P = 0.0001, Table 2). No change was seen after addition of untreated-medium and these samples were not examined for changes in cell dimensions and particle numbers.

Fig. 4.

Fig. 4

Open in a new tab

Effect of Caco-2/macrophage culture time on transepithelial resistance. Delta changes in transepithelial resistance, when compared to control/TER, are calculated as outlined in the section entitled ’Calculation of delta changes in transepithelial resistance‘. Significant differences (P ≤ 0.05) are as follows: #, indicates a significant difference from the Caco-2 group control/TER and different letters (a, b and c) signify a difference in transepithelial resistance change across treatment groups, e.g. ‘b’ under the 12- and 24-h bars indicates that these two values are similar to each other but different from all other time points. Variations in comparator and calculation methods have minimal effect on macrophage-induced changes in Caco-2 transepithelial resistance.

The Caco-2 cells in the Caco-2/macrophage model were less regular in size and shape at all levels, as seen using all antibodies for junctional proteins (Fig. 5). The immunofluorescence labelling of TJ proteins also showed more examples of double lines at apical cell boundaries and/or protein internalisation into the cytoplasm, although these were not a prominent feature. The apical occludin tended to be more diffuse, the ZO-1 labelling often suggested a jagged cell profile and the punctate nature of E-cadherin was less obvious along the lateral wall more basally. The bright, spherical aggregations of junctional protein were still seen. In some areas, large, highly pleomorphic cells were observed, with highly irregular junctional boundaries and 9–29 surrounding cells. Occasionally, these pleomorphic cells lacked a nucleus, but most had no structural abnormalities other than the irregular shape. In approximately two thirds of the Caco-2 cells, it was not possible to distinguish either an apical or a lateral actin layer from that found perinuclearly. Instead the actin staining was present throughout the cytoplasm. Furthermore, around 26 ± 4% of nuclei were located more superficially than those in control samples.

Fig. 5.

Fig. 5

Open in a new tab

Immunofluorescence of Caco-2 cells after compartmentalised culture with macrophages. These cells have been prepared as for Fig. 2 and the columns and rows represent similar views. These cells were not exposed to latex particles. The larger proportion of dark contrast represents greater variation in cell height and as before is not indicative of epithelial discontinuities. In the orthogonal view the arrow marks the level of the picture just above it: although membrane pores are not visible, the z-stacks were always continued down until they were visible. The insert image shows the jagged ZO-1 labelling seen at higher magnifications (×63) and also a clear view of the bright, spherical aggregations of junctional protein. The pattern of junctional staining outlines the irregularities in cell shape and the orthogonal views reflect the reduced cell height: particularly irregular cells described as pleomorphic, are marked with *.

Macrophages alone and the Caco-2/macrophage group produced similarly small amounts of TNF-α and higher amounts of IL-8 (Fig. 3). In contrast, IL-1β and IL-6 were barely detectable in medium from monoculture of either cell type, but were both produced to a substantial extent in the Caco-2/macrophage group.

Exposure of Caco-2 cells to particles alone

Particles suspended in medium formed fewer aggregates (27 ± 3%) than those in water (75 ± 8%, P = 0.0001). The particle/only-5 group showed a marked increase in transepithelial resistance (P = 0.0001) followed by a subsequent decrease from this level to that of the particle/only-60 group (P = 0.0001, Fig. 6). Cells at both particle-only time points were taller and wider than those in the morphology-control group (Table 3) and columnar in shape with no multilayering (Fig. 7A,D), but contained some cellular material within and below the pores of the insert membrane. Confocal microscopy of immunolabelled samples showed that after particle exposure for 60 min, 77 ± 10% of epithelial particles were either on or touching cell junctions, in 10 samples from three experiments (Fig. 7G,H). Sub-membranous particle numbers increased between 5- and 60-min exposure times (P = 0.03, Fig. 8).

Fig. 6.

Fig. 6

Open in a new tab

Transepithelial resistance (TER) of different groups of cells after particle addition. The ‘combined pre-treatment/particle effect’ is (post-particle/TER – control/TER). Significant differences (P ≤ 0.05) are as follows: # indicates a difference from control/TER; P/O-5 or P/O-60 above a histogram bar illustrates a change from the particle/only-5 or -60 groups; M/P-5 and M/P-60 from the macrophage/particle-5 or -60 groups; and solid line shows a difference between particle exposure times, within the same treatment group. Both 24 h ‘combined’ treatments produce a significant TJ opening effect.

Table 3.

Cell dimension measurements.

Cell dimensions in μm (±SEM)
Group Treatment group Height Apical major width Apical minor width Basal major width Basal minor width
A Morphology-control* 27.2 ± 1.7 14.7 ± 0.5 8.7 ± 0.4 10.1 ± 0.4 7.1 ± 0.4
Particle-only group
B Particle/only-5 34.8 ± 0.9A 15.6 ± 0.6 11.8 ± 0.5A 13.3 ± 0.8A 9.1 ± 0.4A
C Particle/only-60 37.2 ± 1.4A 17.5 ± 0.9A 12.2 ± 0.7A 13.6 ± 0.3A 9.9 ± 0.3A
24 h Caco-2/macrophage group
D Macrophage/particle-5 27.5 ± 1.4B 18.5 ± 0.8B 13.4 ± 0.6B 14.5 ± 0.5B 10.6 ± 0.4B
E Macrophage/particle-60 23.9 ± 1.3C 17.0 ± 0.5 14.0 ± 0.5C 15.1 ± 0.5C 11.2 ± 0.4C
24 h macrophage-conditioned medium group
F Macrophage-conditioned medium/particle-5 32.0 ± 2.1D 17.2 ± 0.5B 10.8 ± 0.5D 13.8 ± 0.5 9.0 ± 0.4D
G Macrophage-conditioned medium/particle-60 30.5 ± 1.1CE 17.6 ± 0.6 10.6 ± 0.6E 13.2 ± 0.3E 9.1 ± 0.3E
Open in a new tab

Mean values ± SEM for n = 27 samples are calculated from three independent experiments. *Data for group A are from Moyes et al. (2010). Statistical differences (P ≤ 0.05) between the data for one group and another are indicated by the use of the group letters defined in the first column. Apical widths, both major and minor for all treatment groups, are significantly larger than the equivalent basal dimensions (P = 0.09–0.0001).

Fig. 7.

Fig. 7

Open in a new tab

Confocal analysis of different groups of cells after particle addition. Top panel of confocal microscopy images illustrates Caco-2 cells from particle-only (A and D), Caco-2/macrophage compartmentalised culture (B and E) and macrophage-conditioned medium (C and F) groups after particle exposure for 60 min, stained red with propidium iodide, with fluorescent green microparticles. In the orthogonal view the arrow marks the level of the picture just above it. Schematics of cell dimensions were calculated from a pool of 27 confocal z-stack images for each treatment group (Table 3). After 24 h compartmentalised culture with macrophages and 60 min exposure to particles, the Caco-2 cells are markedly shorter. Lower panel of confocal microscopy images shows particles associated with intercellular junctions (both green) in particle-only samples [occludin (G) or ZO-1 (H)] and after dual culture [occludin (I) and E-cadherin (J)]: all samples were also counterstained with F-actin (red) and DAPI (blue).

Fig. 8.

Fig. 8

Open in a new tab

Particle numbers in the lower well. Histograms (top) illustrate total sub-membranous particle numbers: a log scale is used because of the wide range of numbers across treatment groups. Total sub-membranous counts include wash, lower well fluid (LWF) and macrophage-associated particles (MAP). Significant differences (P≤ 0.05) are given as P/O-5 or P/O-60 as a change from the particle/only-5 or -60 groups, respectively, and M/P-5 and M/P-60 a change from the macrophage/particle-5 or -60 groups. After 24 h Caco-2/macrophage compartmentalised culture, more particles pass through the Caco-2 epithelia to the sub-membranous compartment. Confocal microscopy images (bottom) show lower well macrophages after F-actin labelling (red) at low and high power [×20 (A) or ×63 objective with × 2 digital zoom (B)], the latter also including orthogonal views displaying the z-level at which the apical particle is visible (green). In the orthogonal views the white lines mark the level of the main picture. After particle exposure for 60 min approximately two thirds of macrophage-associated particles in the lower well are either on or within macrophages.

Effect of 24-h macrophage compartmentalised culture on particle–cell interaction

Combined treatment decreased Caco-2 cell transepithelial resistance for both macrophage/particle-5 and -60 groups (P = 0.0001 for both, Fig. 6) below the control/TER level more than the corresponding macrophage-conditioned medium groups (P = 0.0001). The untreated-medium combined response was broadly similar to that of the particle-only groups (Table 2). Isolating the ‘particle effects’ (Table 2) revealed a delta transepithelial resistance increase for the macrophage/particle-5 group (P = 0.0001), although this was smaller than for particle/only-5.

Caco-2 cells in the 24-h Caco-2/macrophage group were shorter and wider at both particle time points (Table 3, Fig. 7B,E) compared with particle-only and macrophage-conditioned medium (Fig. 7C,F) groups, producing a more cuboidal cell. After 60 min particle exposure, many epithelial particles (96 ± 4%) continue to be either on or touching immunolabelled cell junctions, in 8 samples from three experiments (Fig. 7I,J).

More particles passed through the epithelial layer for both the macrophage/particle-5 and -60 groups than for the particle-only (P = 0.02–0.003, Fig. 8) or macrophage-conditioned (P = 0.0001) groups. This was almost entirely because of the many particles associated with the macrophages. On average, after exposure for 60 min, there was one particle to every 154 ± 21 macrophages in the lower well (n = 12 sites from two experiments). Of those associated with macrophages, there were 2 ± 0.4 particles located within the cells to every 1 ± 0.3 found near or on them. Those within a cell were usually at the upper aspect of the cells, i.e. that nearer to the insert (Fig. 8A,B).

Discussion

Although the prime subject of this study is the effect of underlying macrophages on Caco-2 epithelium and particle uptake across it, further comment is first required on the context of the microparticle uptake in this in vitro model compared to the in vivo in situ situation. In the latter, more than 95% of 2 μm particles were identified by microscopy as being taken up through villous epithelium rather than via the follicle-associated epithelium at Peyer's patches (Hodges et al. 1995; Smyth et al. 2008). Furthermore, uptake was also seen in the absence of mucosa-associated lymphoid tissue (Smyth et al. 2008). The in vivo in situ uptake was higher for young adult than younger or older rats, but did not change across species for laboratory animals (Doyle-McCullough et al. 2007). The microparticles also moved onwards in rats to secondary organs such as lymph nodes (LeFevre et al. 1980; Jenkins et al. 1994; McMinn et al. 1996), where they could pose a health risk if toxicologically significant.

These findings had therefore led to a search for the mechanisms involved in microparticle uptake across the mucosal epithelial layer. Transmission electron microscopic examination of thin sections of rat small intestine revealed latex particles not only at the microvillous borders of villous sites and within the blood vessels in the subepithelial stromal tissue, but also in between the epithelial cells (Smyth et al. 2005), particularly in pregnant animals: after processing, the latex particles were of a similar size to the length of the microvilli. The procedure used to select particle-laden areas decreased the quality of the resulting micrographs and made it impossible to detect changes at the TJs, although intercellular spaces containing particles were clearly visible, similar to those observed after other interventions (Madara & Pappenheimer, 1987). However, the identification of intercellular spaces as regions where particles were found made it important to investigate the role in microparticle uptake of the TJs found at the apical boundary of this paracellular compartment. For this, the in vitro Caco-2 monolayer seemed an appropriate model.

The Caco-2 cell response to exposure to 2 μm latex particles in the current experiments shows the typical initial transepithelial resistance increase, which then decreases with time (Moyes et al. 2007, 2008, 2010). The usual increase in particle uptake into the lower well with time and the consistency in cell dimensions confirm that this model makes a good basis for comparison with the further effect of exposure to macrophages or conditioned medium. There are, however, few data from other groups for comparison of the effects of microparticle exposure on Caco-2 TER. Although studies published that are most similar to the current study (McClean et al. 1998; Bravo-Osuna et al. 2008; Gasser et al. 2009; Rekha & Sharma, 2009; Coppi et al. 2010) do not report similar transepithelial resistance changes to those seen here, direct comparisons are not possible due to differences in particle type, size, exposure time and cell model. The effect of temperature on transepithelial resistance (Moyes et al. 2010) also cannot be taken into account, as information on this is not available in all papers. Two new factors support further the suitability of the latex particles as an inert probe in this model: these are the lack of surfactant in the particle preparation as provided by the supplier and the minimal aggregation observed after exposure to the medium used in cell culture. These two aspects of possible input from the particle suspension fluid indicate that it is unlikely to contribute to transepithelial resistance response or particle uptake. Furthermore, previous work has shown that, with respect to the possible effects of dilution of the medium, the addition of 1 μL of fluid as a particle control, using medium and not the water vehicle in which the microparticles are suspended, to the 4 mL already present, also produced an initial transepithelial resistance increase but no subsequent decrease (Moyes et al. 2007). The former can be attributed to the effects of temperature and the earlier transepithelial resistance measurements, rather than solely to the addition of fluid (Moyes et al. 2010).

Confocal microscopy on a morphology-control group of Caco-2 cells (Moyes et al. 2010) showed that the cells were columnar, with polygonal apices and bases: there was also no multilayering above the insert membrane, but cellular material was evident in 60–90% of pores and in some cases as a thin layer below the membrane < 3 μm thick. It is possible that cellular material within the membrane pores would impede the passage of particles through to the lower well, but there is no evidence to support this. The current immunofluorescence images confirm the Caco-2 cell shape of samples stained by propidium iodide (Moyes et al. 2007, 2008) and illustrate the contributions of junctional proteins and actin to the formation of this columnar epithelial sheet with regularly arranged nuclei and cell boundaries. The outline of the cells apically is seen by the labelling of occludin, ZO-1 and the sharply defined E-cadherin line representing the zonula adherens (Ferruzza et al. 1999; Obert et al. 2000; Ma et al. 2004; Musch et al. 2006). On the other hand, the more basal and punctate E-cadherin localisation must be related to the position of desmosomes (Fujimoto, 1995) and the peripheral concentration of actin is similar to that described elsewhere for untreated Caco-2 cells (Shaw et al. 2005). Finally, the application of immunofluorescence to study the effects of microparticle addition to a Caco-2 monolayer shows that, despite the invasive procedures used in preparation of these images, many particles remain and more than two thirds of them are associated with intercellular junctions. This is in line with the earlier reports of intercellular latex particles, using transmission electron microscopy of ultrathin sections of intestinal samples from the in vivo in situ model (Smyth et al. 2005).

The most marked effect of compartmentalised culture with macrophages is the increase in particle uptake across the Caco-2 epithelium, where almost all of the greatly increased numbers of particles are in close proximity to the macrophages. This underlines this cell's role in phagocytosing particles in the lower well, a function which is similar to that of macrophages in vivo. However, this does not necessarily imply that in the in vitro model it would increase the flow of particles from the apical surface of the Caco-2 cells through to the lower well. Other findings in the current paper possibly relevant to the mechanisms involved in the uptake of microparticles are the increased permeability of the Caco-2 monolayer, the role of cytokines and the loss in epithelial height.

With respect to permeability, the extent of the TJ opening induced by Caco-2/macrophage compartmentalised culture in the current paper is very close to the literature value, although there is some divergence at later time points, possibly related to differences in epithelial culture time, control/TER, the exact status of the cells in the lower well or the ratios of epithelial/macrophage cells (Kanzato et al. 2001; Manabe et al. 2003; Satsu et al. 2006). This change in transepithelial resistance is accompanied by a decrease in the regularity of the cells, reflected in the patterns of the junctional proteins. The more diffuse, less apical localisation of the tetraspan TJ protein occludin could be related to a decrease of barrier function, relevant to paracellular particle uptake: it may also be relevant that in this irregular epithelium there remains a high proportion of particles associated with the junctions. The jagged lines related to the ZO-1 immunostaining have also been seen after exposure to TNF-α for 48 h (Ma et al. 2004). This could reflect changes in the Caco-2 TJs, or in the way that ZO-1 interacts with cytoskeletal components such as actin (Fanning et al. 1998, 2002), which is also shown by the current study to be altered by exposure to macrophages. Although there was no apparent change in the E-cadherin labelling at the level of the zonula adherens, the diffuseness of the more basal normally punctate desmosome-related labelling could reflect a weakening of the adhesion between cells, which would allow easier passage of particles through the intercellular space. Another example of irregularities in the intercellular relationships between the Caco-2 cells following compartmentalised culture is the presence of substantial numbers showing pleomorphism in their apical portions.

With respect to the role of cytokines, it has been suggested that proinflammatory cytokines produced by macrophages (Satsu et al. 2006; Al-Sadi et al. 2009) act partly by loosening junctions between Caco-2 cells, thereby increasing access to the sub-epithelial immune cells for both inert and biologically active material present in the intestinal lumen (van der Zanden et al. 2009). Both IL-1β and TNF-α are known separately to increase TJ permeability via myosin light chain kinase-controlled cytoskeletal contraction (Ma et al. 2004; Al-Sadi et al. 2010). Increased levels of IL-1β also decrease occludin expression and disrupt its junctional localisation (Al-Sadi & Ma, 2007) and IL-6 decreases transepithelial resistance by around 50% (Tazuke et al. 2003). This suggests a cytokine control of mechanisms that loosen tight junctions. The addition of both IL-1β and TNF-α also increases Caco-2 IL-8 and -6 secretion (Van De Walle et al. 2010).

Data in the current paper, showing the lack of IL-10 and the low levels of TNF-α, IL-6 and IL-8 produced by Caco-2 cells alone, are in line with a previous report (Parlesak et al. 2004). However, compartmentalised culture leads to higher levels of secretion of IL-1β and IL-6 than are produced by macrophages or Caco-2 cells alone and does not significantly decrease the secretion of TNF-α and IL-8. There is thus little match with the immunosuppressive effect of Caco-2 cells on peripheral blood mononuclear cells exposed to Escherichia coli (Parlesak et al. 2004). The higher levels of IL-1β, -6, -8 and TNF-α produced in the compartmentalised model support the conclusion that cytokine action may contribute to TJ loosening (Tazuke et al. 2003; Ma et al. 2004; Al-Sadi & Ma, 2007; Al-Sadi et al. 2010; Van De Walle et al. 2010). However, mechanisms other than cytokine action must also be important. For example, due to time constraints, the current study does not include data on macrophage-induced Caco-2 cell death, the extent of which has been debated in the literature (Kanzato et al. 2001; Ma et al. 2004; Zhao et al. 2008; Ishimoto et al. 2010): further work could include assays for apoptosis and necrosis.

The results therefore confirm the literature reports of macrophage-induced opening of the Caco-2 TJs and show that this happens at the same time as there is an increase in particle uptake through the epithelial monolayer to the underlying compartment. However, epithelial TJ opening and input from the secretion of cytokines are not the only factors associated with increased microparticle uptake through a Caco-2 monolayer exposed in compartmentalised culture to medium in contact with underlying macrophages. Another possible factor is the macrophage-induced decrease in epithelial height, which must reduce the distance microparticles are required to travel between the delivery point at the cell apex and the fluid on the basal side of the epithelium. This change in height may be the result of remodelling of the actin component of the cytoskeleton, evident in the overall reduction in the supranuclear actin-free region described above for untreated Caco-2 cells. However, intracytoplasmic changes in the cytoskeleton will only have an indirect effect on paracellular particle passage through the intercellular spaces. Although there is no further evidence that the shorter epithelium seen after compartmentalised culture is causally linked to the increased microparticle uptake, the possibility that this is the case is supported by the unchanged cell height and lack of increased sub-membranous particle numbers in samples treated only with macrophage-conditioned medium.

In conclusion, the exposure of the Caco-2 cells to macrophages increases particle uptake across the epithelial monolayer and into the subepithelial compartment within the same time frame as the loosening of the epithelial TJs and the reduction of cell height.

Acknowledgments

We are grateful to the Department of Health's Radiation Protection Research Program for financial support. The authors would also like to thank the following people; Professor Dame K. E. Davies for the use of cell culture facilities; Drs C. Norbury and K. Gibson for advice on the actin immunofluorescence labelling; Dr C. A. R. Boyd for advice on aspects of the text revision; the Department of Statistics for advice on data analysis; Mrs S. Newton at the University of Sheffield for the cytokine analysis; Dr J. Runions and Professor C. Hawes at Oxford Brookes University for their advice and access to the Zeiss LSM510 confocal microscope; and colleagues in the Department of Physiology, Anatomy and Genetics for access to the Zeiss LSM710 confocal microscope.

References

  1. Al-Sadi RM, Ma TY. IL-1beta causes an increase in intestinal epithelial tight junction permeability. J Immunol. 2007;178:4641–4649. doi: 10.4049/jimmunol.178.7.4641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Al-Sadi R, Boivin M, Ma T. Mechanism of cytokine modulation of epithelial tight junction barrier. Front Biosci. 2009;14:2765–2778. doi: 10.2741/3413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Al-Sadi R, Dongmei Y, Hamid MS, et al. Cellular and molecular mechanism of interleukin-1B modulation of CACO-2 intestinal epithelial tight junction barrier. J Cell Mol Med. 2010 doi: 10.1111/j.1582-4934.2010.01065.x. DOI: 10.1111/j.1582-4934.2010.01065.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bravo-Osuna I, Vauthier C, Chacun H, et al. Specific permeability modulation of intestinal paracellular pathway by chitosan-poly(isobutylcyanoacrylate) core-shell nanoparticles. Eur J Pharm Biopharm. 2008;69:436–444. doi: 10.1016/j.ejpb.2007.12.012. [DOI] [PubMed] [Google Scholar]
  5. Carr KE, Hazzard RA, Reid S, et al. The effect of size on uptake of orally administered latex microparticles in the small intestine and transport to mesenteric lymph nodes. Pharm Res. 1996;13:1205–1209. doi: 10.1023/a:1016064320334. [DOI] [PubMed] [Google Scholar]
  6. Coppi G, Montanari M, Rossi T, et al. Cellular uptake and toxicity of microparticles in a perspective of polymyxin B oral administration. Int J Pharm. 2010;385:42–46. doi: 10.1016/j.ijpharm.2009.10.026. [DOI] [PubMed] [Google Scholar]
  7. Cuddihy RG, Finch GL, Newton GJ, et al. Characteristics of radioactive particles released from the Chernobyl nuclear reactor. Environ Sci Technol. 1989;23:89–95. [Google Scholar]
  8. Delie F, Rubas W. A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model. Crit Rev Ther Drug Carrier Syst. 1997;14:221–286. [PubMed] [Google Scholar]
  9. Doyle-McCullough M, Smyth SH, Moyes SM, et al. Factors influencing intestinal microparticle uptake in vivo. Int J Pharm. 2007;335:79–89. doi: 10.1016/j.ijpharm.2006.10.043. [DOI] [PubMed] [Google Scholar]
  10. Ebel JP. A method for quantifying particle absorption from the small intestine of the mouse. Pharm Res. 1990;7:848–851. doi: 10.1023/a:1015964916486. [DOI] [PubMed] [Google Scholar]
  11. Fanning AS, Jameson BJ, Jesaitis LA, et al. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem. 1998;273:29745–29753. doi: 10.1074/jbc.273.45.29745. [DOI] [PubMed] [Google Scholar]
  12. Fanning AS, Ma TY, Anderson JM. Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1. FASEB J. 2002;16:1835–1837. doi: 10.1096/fj.02-0121fje. [DOI] [PubMed] [Google Scholar]
  13. Ferruzza S, Scarino ML, Rotilio G, et al. Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells. Am J Physiol. 1999;277:G1138–G1148. doi: 10.1152/ajpgi.1999.277.6.G1138. [DOI] [PubMed] [Google Scholar]
  14. Florence AT. The oral absorption of micro- and nanoparticulates: neither exceptional nor unusual. Pharm Res. 1997;14:259–266. doi: 10.1023/a:1012029517394. [DOI] [PubMed] [Google Scholar]
  15. Fujimoto K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. J Cell Sci. 1995;108(Pt 11):3443–3449. doi: 10.1242/jcs.108.11.3443. [DOI] [PubMed] [Google Scholar]
  16. Gasser M, Riediker M, Mueller L, et al. Toxic effects of brake wear particles on epithelial lung cells in vitro. Part Fibre Toxicol. 2009;6:30. doi: 10.1186/1743-8977-6-30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hallab NJ, Jacobs JJ. Biologic effects of implant debris. Bull NYU Hosp Jt Dis. 2009;67:182–188. [PubMed] [Google Scholar]
  18. Hodges GM, Carr EA, Hazzard RA, et al. Uptake and translocation of microparticles in small intestine. Morphology and quantification of particle distribution. Dig Dis Sci. 1995;40:967–975. doi: 10.1007/BF02064184. [DOI] [PubMed] [Google Scholar]
  19. Hodgson A, Scott JE, Fell TP, et al. Doses from the consumption of Cardiff Bay flounder containing organically bound tritium. J Radiol Prot. 2005;25:149–159. doi: 10.1088/0952-4746/25/2/003. [DOI] [PubMed] [Google Scholar]
  20. Inoue M, Fujii H, Kaseyama H, et al. Stimulation of macrophages by mucins through a macrophage scavenger receptor. Biochem Biophys Res Commun. 1999;264:276–280. doi: 10.1006/bbrc.1999.1515. [DOI] [PubMed] [Google Scholar]
  21. Ishimoto Y, Nakai Y, Satsu H, et al. Transient up-regulation of immunity- and apoptosis-related genes in Caco-2 cells cocultured with THP-1 cells evaluated by DNA microarray analysis. Biosci Biotechnol Biochem. 2010;74:437–439. doi: 10.1271/bbb.90732. [DOI] [PubMed] [Google Scholar]
  22. Jani P, Halbert GW, Langridge J, et al. Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency. J Pharm Pharmacol. 1990;42:821–826. doi: 10.1111/j.2042-7158.1990.tb07033.x. [DOI] [PubMed] [Google Scholar]
  23. Jenkins AP, Menzies IS, Nukajam WS, et al. The effect of ingested lactulose on absorption of L-rhamnose, D-xylose, and 3-O-methyl-D-glucose in subjects with ileostomies. Scand J Gastroenterol. 1994;29:820–825. doi: 10.3109/00365529409092517. [DOI] [PubMed] [Google Scholar]
  24. Kanzato H, Manabe M, Shimizu M. An in vitro approach to the evaluation of the cross talk between intestinal epithelium and macrophages. Biosci Biotechnol Biochem. 2001;65:449–451. doi: 10.1271/bbb.65.449. [DOI] [PubMed] [Google Scholar]
  25. LeFevre ME, Hancock DC, Joel DD. Intestinal barrier to large particulates in mice. J Toxicol Environ Health. 1980;6:691–704. doi: 10.1080/15287398009529888. [DOI] [PubMed] [Google Scholar]
  26. Ma TY, Iwamoto GK, Hoa NT, et al. TNF-α-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation 10.1152/ajpgi.00173.2003. Am J Physiol Gastrointest Liver Physiol. 2004;286:G367–G376. doi: 10.1152/ajpgi.00173.2003. [DOI] [PubMed] [Google Scholar]
  27. Madara JL, Pappenheimer JR. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia. J Membr Biol. 1987;100:149–164. doi: 10.1007/BF02209147. [DOI] [PubMed] [Google Scholar]
  28. Manabe M, Takenaka R, Nakasa T, et al. Induction of anti-inflammatory responses by dietary Momordica charantia L. (bitter gourd) Biosci Biotechnol Biochem. 2003;67:2512–2517. doi: 10.1271/bbb.67.2512. [DOI] [PubMed] [Google Scholar]
  29. McClean S, Prosser E, Meehan E, et al. Binding and uptake of biodegradable poly-DL-lactide micro- and nanoparticles in intestinal epithelia. Eur J Pharm Sci. 1998;6:153–163. doi: 10.1016/s0928-0987(97)10007-0. [DOI] [PubMed] [Google Scholar]
  30. McMinn LH, Hodges GM, Carr KE. Gastrointestinal uptake and translocation of microparticles in the streptozotocin-diabetic rat. J Anat. 1996;189(Pt 3):553–559. [PMC free article] [PubMed] [Google Scholar]
  31. Moyes SM, Smyth SH, Shipman A, et al. Parameters influencing intestinal epithelial permeability and microparticle uptake in vitro. Int J Pharm. 2007;337:133–141. doi: 10.1016/j.ijpharm.2006.12.036. [DOI] [PubMed] [Google Scholar]
  32. Moyes SM, Killick EM, Morris JF, et al. Changes produced by external radiation in parameters influencing intestinal permeability and microparticle uptake in vitro. Int J Radiat Biol. 2008;84:467–486. doi: 10.1080/09553000802078388. [DOI] [PubMed] [Google Scholar]
  33. Moyes SM, Morris JF, Carr KE. Culture conditions and treatments affect Caco-2 characteristics and particle uptake. Int J Pharm. 2010;387:7–18. doi: 10.1016/j.ijpharm.2009.11.027. [DOI] [PubMed] [Google Scholar]
  34. Musch MW, Walsh-Reitz MM, Chang EB. Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointest Liver Physiol. 2006;290:G222–G231. doi: 10.1152/ajpgi.00301.2005. [DOI] [PubMed] [Google Scholar]
  35. Obert G, Peiffer I, Servin AL. Rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal Caco-2 cell monolayers. J Virol. 2000;74:4645–4651. doi: 10.1128/jvi.74.10.4645-4651.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parlesak A, Haller D, Brinz S, et al. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria. Scand J Immunol. 2004;60:477–485. doi: 10.1111/j.0300-9475.2004.01495.x. [DOI] [PubMed] [Google Scholar]
  37. Powell JJ, Faria N, Thomas-McKay E, et al. Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract. J Autoimmun. 2010;34:J226–J233. doi: 10.1016/j.jaut.2009.11.006. [DOI] [PubMed] [Google Scholar]
  38. Rekha MR, Sharma CP. Synthesis and evaluation of lauryl succinyl chitosan particles towards oral insulin delivery and absorption. J Control Release. 2009;135:144–151. doi: 10.1016/j.jconrel.2009.01.011. [DOI] [PubMed] [Google Scholar]
  39. Rescigno M, Urbano M, Valzasina B, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol. 2001;2:361–367. doi: 10.1038/86373. [DOI] [PubMed] [Google Scholar]
  40. Satsu H, Ishimoto Y, Nakano T, et al. Induction by activated macrophage-like THP-1 cells of apoptotic and necrotic cell death in intestinal epithelial Caco-2 monolayers via tumor necrosis factor-alpha. Exp Cell Res. 2006;312:3909–3919. doi: 10.1016/j.yexcr.2006.08.018. [DOI] [PubMed] [Google Scholar]
  41. Shaw RK, Smollett K, Cleary J, et al. Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells. Infect Immun. 2005;73:4385–4390. doi: 10.1128/IAI.73.7.4385-4390.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shi SR, Chaiwun B, Young L, et al. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993;41:1599–1604. doi: 10.1177/41.11.7691930. [DOI] [PubMed] [Google Scholar]
  43. Smyth SH, Doyle-McCullough M, Cox OT, et al. Effect of reproductive status on uptake of latex microparticles in rat small intestine. Life Sci. 2005;77:3287–3305. doi: 10.1016/j.lfs.2005.04.030. [DOI] [PubMed] [Google Scholar]
  44. Smyth SH, Feldhaus S, Schumacher U, et al. Uptake of inert microparticles in normal and immune deficient mice. Int J Pharm. 2008;346:109–118. doi: 10.1016/j.ijpharm.2007.06.049. [DOI] [PubMed] [Google Scholar]
  45. Stather JW. The polonium-210 poisoning in London. J Radiol Prot. 2007;27:1–3. doi: 10.1088/0952-4746/27/1/E02. [DOI] [PubMed] [Google Scholar]
  46. Tazuke Y, Drongowski RA, Teitelbaum DH, et al. Interleukin-6 changes tight junction permeability and intracellular phospholipid content in a human enterocyte cell culture model. Pediatr Surg Int. 2003;19:321–325. doi: 10.1007/s00383-003-1003-8. [DOI] [PubMed] [Google Scholar]
  47. Van De Walle J, Hendrickx A, Romier B, et al. Inflammatory parameters in Caco-2 cells: effect of stimuli nature, concentration, combination and cell differentiation. Toxicol In Vitro. 2010;24:1441–1449. doi: 10.1016/j.tiv.2010.04.002. [DOI] [PubMed] [Google Scholar]
  48. Wells CL, Maddaus MA, Erlandsen SL, et al. Evidence for the phagocytic transport of intestinal particles in dogs and rats. Infect Immun. 1988;56:278–282. doi: 10.1128/iai.56.1.278-282.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wells CL, van de Westerlo EM, Jechorek RP, et al. Cytochalasin-induced actin disruption of polarized enterocytes can augment internalization of bacteria. Infect Immun. 1998;66:2410–2419. doi: 10.1128/iai.66.6.2410-2419.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. van der Zanden EP, Snoek SA, Heinsbroek SE, et al. Vagus nerve activity augments intestinal macrophage phagocytosis via nicotinic acetylcholine receptor alpha4beta2. Gastroenterology. 2009;137:1029–1039. doi: 10.1053/j.gastro.2009.04.057. [DOI] [PubMed] [Google Scholar]
  51. Zhao Z, Satsu H, Fujisawa M, et al. Attenuation by dietary taurine of dextran sulfate sodium-induced colitis in mice and of THP-1-induced damage to intestinal Caco-2 cell monolayers. Amino Acids. 2008;35:217–224. doi: 10.1007/s00726-007-0562-8. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Anatomy are provided here courtesy of Anatomical Society of Great Britain and Ireland

RESOURCES