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. 2003 Dec;110(4):466–473. doi: 10.1111/j.1365-2567.2003.01759.x

DEC-205lo Langerinlo neonatal Langerhans' cells preferentially utilize a wortmannin-sensitive, fluid-phase pathway to internalize exogenous antigen

Bernadette M Bellette 1, Gregory M Woods 1, Teresa Wozniak 1, Kathleen V Doherty 1, H Konrad Muller 1
PMCID: PMC1783083  PMID: 14632644

Abstract

Antigen treatment of neonatal epidermis results in antigen-specific immune suppression. Compared with adult counterparts, neonatal Langerhans' cells (LC) demonstrate an impaired ability to transport antigen to the lymph node (LN). As it is possible that neonatal LC have a reduced ability to endocytose antigen, we evaluated the acquisition of endocytic function, the expression of uptake receptors and the internalization of soluble and small particulate antigens in neonatal, juvenile and adult mice. Although LC from 4-day-old mice were weakly positive for the mannose-type receptor, Langerin, they were capable of internalizing fluorescein isothiocyanate (FITC)-dextran, but to a lesser extent than LC from 6-week-old mice. However, when ratio data were calculated to account for variations in fluorescence intensity at 4°, it was demonstrated that neonatal LC continued to internalize antigen over a longer period of time than adult mice and, as the ratios were much higher, that neonatal cells were also relatively more efficient in antigen uptake. When receptors for mannan and mannose were competitively blocked, LC from neonatal mice, but not adult mice, could still efficiently internalize FITC–dextran. Consequently, the uptake of FITC–dextran, in part, occurred via alternative receptors or a receptor-independent fluid-phase pathway. A feasible pathway is macropinocytosis, as LC from 4-day-old mice demonstrated a reduction in FITC–dextran internalization by the macropinocytosis inhibitor, wortmannin. Evidence of a functional macropinocytosis pathway in neonatal LC was further supported by internalization of the soluble tracer Lucifer Yellow (LY). We conclude that neonatal LC preferentially utilize a wortmannin-sensitive, fluid-phase pathway, rather than receptor-mediated endocytosis, to internalize antigen. As neonatal LC are capable of sampling their environment without inducing immunity, this may serve to avoid inappropriate immune responses during the neonatal period.

Introduction

Langerhans' cells (LC) belong to the dendritic cell family and, as the sentinel antigen-presenting cell of the epidermis, are pivotal to the initiation of cutaneous immune responses.1 LC exist in two distinct forms, governed by their location and function.2 When resident in the epidermis, LC are primarily involved in antigen uptake and processing, sampling their external environment via fluid-phase pinocytosis and endocytic receptors.3,4 Following uptake, LC process the antigen and, after detachment from neighbouring keratinocytes, migrate via the afferent lymphatics to the draining lymph node (LN).5 During transit, LC undergo a process of differentiation so that their function converts to an ability to trigger the activation of naïve T cells.6 This change in function is reflected by a phenotypic alteration, including the up-regulation of major histocompatibility complex class II (MHC class II)7 and of the costimulatory molecules CD40,8,9 CD80 and CD86.10,11 Fundamental to the initiation of an immune response is successful antigen uptake, followed by the up-regulation of costimulatory molecules.

LC are capable of employing an array of uptake pathways that accommodate both small and large particulate antigens, as well as soluble peptides.12 Receptor-mediated endocytosis involves the internalization of small particulate antigen following the recognition and binding of membrane components by uptake receptors.13 Receptors expressed by LC include the lectin-type receptors DEC-20514,15 and Langerin16 (implicated in the uptake of carbohydrate-conjugated antigen), the Fc receptors CD16 and CD32,17,18 and the complement receptors CR3 and CR4.19 Large particulate antigens, such as microbial pathogens, can be engulfed by phagocytosis,20 a process that utilizes receptors for prokaryotic peptides, including lipopolysaccharide21 and peptidoglycan.22 Following receptor binding and antigen internalization, a cascade of events is initiated that leads to augmentation of the immune response by inducing secretion of pro-inflammatory cytokines and up-regulation of costimulatory molecules.23

Soluble antigen is internalized in a non-specific manner by pinocytosis, which occurs via two distinct mechanisms.24 Small molecules (≈ 0·1 µm) are internalized via clathrin-coated pits (micropinocytosis). Uptake of larger vesicles (0·5–3 µm) is mediated by cytoskeleton-dependent membrane ruffling (macropinocytosis).24 Although many cell types perform macropinocytosis, dendritic cells are the only cell type to pinocytose constitutively without the receipt of external stimuli.25 This mechanism enables the cell to engulf a large volume of fluid, from which soluble antigens are then concentrated for processing and presentation.26

We have previously demonstrated that LC from 3-day-old mice lack expression of the mannose-binding receptor DEC-205 and exhibit a reduction in dendrite formation.27 This led to the conclusion that during the early neonatal period the capacity of LC to uptake antigen via a receptor-mediated pathway was reduced. The possibility that neonatal LC had a decreased ability to effectively internalize cutaneous antigen was further supported when, following application of antigen through 3-day-old epidermis, the amount of antigen transported by LC to the draining LN was reduced compared with adult counterparts.27 As the amount of antigen presented in the context of MHC class II can influence the outcome of T-cell activation,28 the divergence in the pathways utilized by neonatal LC to internalize and process antigen has important implications for the generation of immune suppression. To assess the acquisition of endocytic function during development, the expression of antigen-binding molecules, and the in vitro uptake of soluble and small particulate antigens, was assessed in neonatal, juvenile and adult mice.

Materials and methods

Animals

Adult (6–12 weeks old), juvenile (7 and 14 days old) and neonatal (4 days old) male and female BALB/c (H-2d) mice were obtained from the University of Tasmania Central Animal House. Animals were used in accordance with the University of Tasmania Ethics Committee (permit numbers 5519, A5620, 98013 and 6063).

Epidermal sheets

Six-week-old and 14-day-old mice were killed by carbon dioxide inhalation, and 7- and 4-day-old mice were anaesthetized for 5 min at −20° followed by decapitation. Dorsal fur was removed with animal clippers and depilatory cream (Veet; Reckitt and Colman, Sydney, Australia). Skin was thoroughly washed with warm tap water and the keratin layer removed with light tape stripping. Fresh tape was then applied and the skin excised into 1-cm2 segments. Skin segments were then incubated, dermis-side down, in phosphate-buffered saline (PBS) containing 20 mm EDTA, for 2 hr at 37°. The epidermis/tape sections were then mechanically removed from the underlying dermis. The resulting sheets were fixed for 10 min in acetone and washed three times in PBS. Sheets were incubated in anti-mouse Langerin (Clone DCGM4, kindly provided by S. Saeland, Schering Plough, Dardilly, France), for 30 min at 37° followed by overnight incubation at 4°. Epidermal sheets were washed three times in PBS and then incubated in a secondary antibody-fluorescein conjugate for 2 hr at 37°[anti-mouse immunoglobin G (IgG) fluorescein isothiocyanate (FITC) conjugate, F(ab′)2 fragment; Cat. no. 985051020, Silenus, Victoria, Australia]. Epidermal sheets were washed three times in PBS and mounted in DAKO mounting media (Cat. no. 002627; DAKO, Carpentaria, CA). Sheets were stored at 4°.

Epidermal suspensions

Adult, juvenile and neonatal mice were killed and their fur removed with clippers and depilatory cream (Veet; Reckitt and Colman). Skin was thoroughly washed with warm tap water, excised and cut into 1-cm2 sections, and then incubated in Hanks' balanced salt solution (HBSS; Invitrogen, Melbourne, Australia), supplemented with 0·5% dispase (Invitrogen) and 100 U/ml DNAse (Invitrogen), for 2 hr at 37°, with slight agitation. Six-week-old and 14-day-old epidermis were mechanically separated from the dermis by scraping with a small spatula, or for 4- and 7-day-old epidermis, gently peeled with fine forceps. The resulting epidermal scrapings were incubated in HBSS (containing 0·5% dispase and 100 U/ml DNAse) for 40 min at 37°, with slight agitation. Following this, an equal volume of RPMI-1640 supplemented with 10% fetal calf serum (FCS; CSL Biosciences, Melbourne Australia), 30 µg/ml gentamicin (David Bull Laboratories, Melbourne, Australia), 2 mm l-glutamate (CSL Biosciences), 1 mm sodium pyruvate (Invitrogen) and 5 × 10−5 m 2-mercaptoethanol (2-ME; Sigma, St Louis, MO), was added and swirled at room temperature for 5 min. To remove particulate matter, the epidermal preparation was filtered through cotton wool and a 40-µm filter, and centrifuged twice at 400 g for 5 min in complete RPMI-1640 containing 10% FCS. After a final wash with RPMI-1640 containing 10% FCS, the pellet was resuspended in 1 ml of RPMI-1640 containing 10% FCS.

Flow cytometry

To determine the level of CD16/CD32 expression, ≈5 × 105 epidermal cells were incubated at 4° for 30 min with purified anti-mouse CD16/32 (FcγIII/II) (Clone 2.4G2, Cat. no. 553142; PharMingen, San Diego, CA). Following incubation, cells were washed three times in PBS containing 5% FCS and 0·1% sodium azide. After washing, cells were incubated in a secondary antibody-fluorescein conjugate (anti-rat IgG FITC conjugate, Cat. no. 984321020; Silenus) for 30 min at 4°. After three washes, cells were incubated for 30 min at 4° with PE rat-anti-mouse I-A/I-E (Clone M5/114·15·2, Cat. no. 557000; PharMingen) and 7-aminoactinomycin D (7-AAD, Cat. No. A 9400; Sigma) and incubated at 4° for 30 min. After three washes, the fluorescence intensity of epidermal cell suspensions was analysed using a Coulter ELITE ESP flow cytometer (Coulter, Los Angeles, CA) equipped with a Coherent Innova 90 argon ion laser.

To determine the total Langerin expression, cells were first incubated with PE rat-antimouse I-A/I-E and 7-AAD, washed and fixed in 4% paraformaldehyde at room temperature for 10 min. Following two washes in 0·1% saponin buffer, cells were incubated for 30 min at 4° with purified anti-Langerin in the presence of saponin. After three washes, the cells were incubated at 4° with a secondary antibody-FITC conjugate (anti-mouse immunoglobulin fluorescein conjugate, Cat. no. 985051020; Silenus) for 30 min. The cells were washed once in saponin buffer, once in PBS and then refixed in 4% paraformaldehyde. Expression level was then quantified via flow cytometry. For all antibody-staining experiments, isotype-matched controls were run in parallel.

Endocytic and macropinocytic antigens

On the day of use, FITC–dextran (ICN Biomedicals, Aurora, OH) and Lucifer Yellow (LY; Sigma) were reconstituted to 2 mg/ml in complete RPMI-1640/FCS and vortexed gently to remove aggregates.

Endocytic assays

Cell suspensions were pulsed with antigen or fluorescent particles at 37° and 4°. The 4° control was included to determine the degree of non-specific surface binding of the antigen to cells. Following incubation, uptake was ceased by the addition of ice-cold PBS containing 5% FCS and 0·1% sodium azide and the suspensions were then washed three times by pulse spinning, in an Eppendorf centrifuge 5415D, for 10 seconds. Epidermal suspensions were stained with PE rat-anti-mouse I-A/I-E (Clone M5/114·15·2, Cat. no. 557000; PharMingen) and 7-AAD (Sigma) for 30 min at 4°. After three washes in PBS containing 5% FCS and 0·1% sodium azide, cells were analysed by flow cytometry.

For blocking of mannose-type receptors, epidermal suspensions were preincubated with mannan from Saccharomyces cerevisiae (Cat. no. M-7504; Sigma) and D(+)-mannose (Cat. No. M-6020; Sigma), at a final concentration of 10 mg/ml, for 10 min at 37° and 4°. Cells were then transferred to 37° and incubated with 1 mg/ml of FITC–dextran for 15 min in the continuous presence of sugars. To inhibit macropinocytosis, epidermal suspensions were incubated with 10 µm of the phosphatidylinositol 3-kinase inhibitor wortmannin, from Penicillium fumiculosum (Cat. No. W-1628, Sigma), for 30 min at 37° followed by incubation with FITC–dextran for 15 min in the continuous presence of inhibitor.

Analysis

Using cellquest™ software, version 3.2.1 (Becton Dickinson, San Jose, CA), dead cells (7-AAD+) were excluded and LC identified on the basis of MHC class II staining. To assess the rate of uptake and amount of antigen internalized during uptake assays, the mean fluorescence intensity at 37° was divided by the mean fluorescence intensity at 4°. This normalization procedure was crucial as it enabled comparison of neonatal, juvenile and adult LC uptake assays, which were performed on separate days.

Results

Identification of LC in epidermal suspensions

In order to select for viable cells, a gate was drawn around the 7-AAD− population. To identify LC in cell suspensions during development, PE-conjugated anti-MHC class II was used. As shown in Fig. 1, a discrete population of viable MHC class II bright (MHC class IIhi) cells were present at each stage of development.

Figure 1.

Figure 1

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Identification of Langerhans' cells (LC) in epidermal suspensions. A gating strategy is shown for the identification of LC in epidermal suspensions. Epidermal suspensions were prepared from murine epidermis by digestion with dispase at 4 days (top panel), 7 days (second top panel), 14 days (second bottom panel) and 6 weeks (bottom panel) and then stained with anti-major histocompatibility complex class II (MHC class II). To select for viable cells, a gate was first drawn around 7-aminoactinomycin D-negative (7-AAD−) cells (data not shown). LC are represented by the discrete population of viable MHC class II+ cells (data are representative of three or more experiments).

Analysis of Langerin expression during neonatal development

Our previous investigations on epidermal sheets demonstrated that expression of the type II lectin, DEC-205, was absent on day 3 following birth but increased with maturity.27 To assess expression of mannose-type lectin receptors, epidermal sheets and suspensions were prepared from 4-, 7- and 14-day-old and 6-week-old mice and stained for expression of the lectin receptor, Langerin. As shown in Fig. 2(a), a gradual acquisition of Langerin expression occurred throughout development. Langerin-positive cells were identified 4 days following birth, with faint staining concentrated to the cell body and around the nucleus. On day 7 following birth the labelling had extended to the cell body periphery. By day 14 following birth, the antigen expression had intensified and was equivalent to that within adult epidermis, with staining distributed throughout the cell, including the cell body, nucleus and dendritic processes. To further quantify the level of Langerin expression, epidermal cell suspensions from neonatal, juvenile and adult mice were permeabilized, stained and evaluated via flow cytometry. As shown in Fig. 2(b), the fluorescence intensity of Langerin staining increased with age of the animal, with very low levels detected 4 days following birth and increasing with maturation.

Figure 2.

Figure 2

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Expression of Langerin by neonatal, juvenile and adult Langerhans' cells (LC). Expression of the mannose-binding receptor Langerin is shown on cells from neonatal, juvenile and adult epidermis. Epidermal sheets were prepared from mice at 4 days (top panel), 7 days (second top panel), 14 days (second bottom panel) and 6 weeks (bottom panel), acetone fixed and then stained for Langerin expression (a). Langerin expression was initially weak and expression gradually increased during maturation (data are representative of five separate experiments). To quantify the level of Langerin expression, epidermal suspensions were prepared from mice at 4 days (top panel), 7 days (second top panel), 14 days (second bottom panel) and 6 weeks (bottom panel), permeabilized and stained for Langerin expression (b). Langerin expression was initially negative and expression gradually increased during maturation. The solid histogram shows the profile of binding of the anti-Langerin antibody. The dotted histogram shows the degree of binding of the isotype-matched control antibody. The mean fluorescence intensity of anti-Langerin staining is shown in parenthesis at the top right hand corner of each histogram (data are representative of two separate experiments).

Analysis of IgG receptor expression (CD16/CD32) during neonatal development

The potential of LC to bind antigen during the early neonatal period was investigated by evaluating the expression of the IgG receptor CD16/CD32. This receptor is important in the recognition of antigen–antibody complexes.

Analysis of cell suspensions via flow cytometry demonstrated that CD16/CD32 was present on day 4 and that the level of expression remained constant during development, although adult LC had the highest level (Fig. 3).

Figure 3.

Figure 3

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Expression of CD16/CD32 by neonatal, juvenile and adult Langerhans' cells (LC). Epidermal suspensions were prepared from mice at 4 days (top panel), 7 days (second top panel), 14 days (second bottom panel) and 6 weeks (bottom panel), and double stained for the expression of CD16/CD32 and major histocompatibility complex class II (MHC class II). Fluorescence was analysed via flow cytometry and further analysed using cellquest software. The solid histogram shows the binding profile of anti-CD16/CD32. The dotted histogram shows the degree of binding of the isotype-matched control antibody. The mean fluorescence intensity of anti-CD16/CD32 staining is shown in parenthesis at the top right hand corner of each histogram (data are representative of three separate experiments).

Uptake of FITC–dextran by neonatal, juvenile and adult LC

The limited expression of Langerin on LC from 4-day-old mice suggests that during the early neonatal period, LC are deficient in their ability to bind and internalize mannose-conjugated antigen via receptor-mediated endocytosis. This hypothesis was first investigated by evaluating the capacity of LC (isolated from day 4 and adult epidermis) to internalize FITC–dextran, a mannosylated antigen believed to be internalized via the mannose receptor to which DEC-205 is homologous. As shown in Fig. 4(a), LC from 4-day-old and 6-week-old epidermis accumulated FITC–dextran, as demonstrated by a time-dependent increase in the FITC–dextran fluorescence intensity at 37°. However, adult LC demonstrated a greater fluorescence intensity at 4°, suggestive of a higher degree of binding of antigen in a non-metabolically active manner, either non-specifically or to cell-surface or membrane receptors. To account for these variations at 4°, the fluorescence intensity at 37° was divided by the intensity of the sample at 4°, generating a ratio for day 4 and adult LC at each time-point (Fig. 4b). To directly compare the antigen uptake at different ages, the data were calculated as a ratio for each time-point (Fig. 4c). Although the initial rate of uptake was similar between neonatal and adult LC, neonatal LC continued to internalize antigen over a longer period of time. It can also be seen from the ratio data that the end-point of antigen uptake is reduced with the age, as LC from 4- and 7-day-old mice reached a higher ratio value than 14-day-old and 6-week-old mice.

Figure 4.

Figure 4

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Uptake of fluorescein isothiocyanate (FITC)–dextran by neonatal, juvenile and adult Langerhans' cells (LC). In vitro uptake of FITC–dextran via receptor-mediated endocytosis at 37° and 4° was assessed in epidermal cells isolated from mice at 4 days and 6 weeks following birth (a). To account for any variations in non-specific or receptor binding of the antigen at 4°, the mean fluorescence intensity at 37° was divided by the mean fluorescence intensity at 4°, generating a ratio for day 4 and adult LC at each time-point (b). To directly compare differences between the different age points, ratio data were then calculated for each age point for each time point. As shown in (c), LC from 4-day-old mice took longer to reach maximum uptake compared with adult LC (each value is representative of three separate experiments).

Effect of mannan, mannose and wortmannin on the uptake of FITC–dextran by neonatal, juvenile and adult LC

As LC isolated from 4-day-old epidermis are highly efficient at internalizing FITC–dextran, it is possible that neonatal LC utilize receptors other than DEC-205 and Langerin, or use a receptor-independent mechanism, to internalize mannosylated antigen. To determine the pathway employed to internalize FITC–dextran, LC from 4-day-old and adult mice were preincubated with the bacterial polysaccharides, mannan and mannose, to inhibit the receptor-mediated endocytic pathway and with the phosphatidylinositol 3-kinase inhibitor, wortmannin, to inhibit the macropinocytosis pathway. As shown in Fig. 5, it is evident that FITC–dextran uptake by LC from 6-week-old mice is inhibited by mannose and mannan to a greater extent than LC from 4-day-old mice. In contrast, wortmannin inhibited macropinocytosis to a greater extent in LC from 4-day-old mice than in LC from 6-week-old mice. Consequently, neonatal LC rely on macropinocytosis more than endocytosis. In contrast, adult LC rely on endocytosis to a greater extent than macropinocytosis.

Figure 5.

Figure 5

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Effect of mannan, mannose and wortmannin on the uptake of fluorescein isothiocyanate (FITC)–dextran by neonatal, juvenile and adult Langerhans' cells (LC). The results show the uptake of FITC–dextran (in the continuous presence of mannan, mannose and wortmannin) by epidermal cells isolated from mice at 4 days and 6 weeks following birth. Cells were preincubated with inhibitors at either 37° or 4° prior to incubation with FITC–dextran at 37°. The percentage inhibition in uptake was calculated as the percentage decrease of FITC–dextran mean fluorescence intensity at 37°, in the presence of inhibitors, compared with cells that were not preincubated with inhibitors (data are representative of two separate experiments).

Uptake of LY by neonatal, juvenile and adult LC

The ability of LC to internalize antigen via macropinocytosis during the early neonatal period was first investigated by evaluating the capacity of LC isolated from 4-day-old and 6-week-old epidermis to internalize LY, a soluble tracer known to be a definitive marker of macropinocytosis. As shown in Fig. 6(a), LC from 4-day-old and 6-week-old epidermis accumulated LY, as demonstrated by a time-dependent increase in LY fluorescence intensity at 37°, However, similarly to FITC–dextran accumulation assays, adult LC demonstrated a much higher degree of non-specific or non-metabolically active binding of antigen at 4°. When ratio data were calculated to account for these variations, it was found that although the initial rate of uptake was similar between neonatal and adult LC, neonatal LC internalized antigen over a longer period of time (Fig. 6b). It is also apparent that there was a gradual decrease in the end-point of antigen uptake with age of the animal, as LC from 4- and 7-day-old mice reached a higher ratio value than LC from 14-day-old and 6-week-old mice (Fig. 6c).

Figure 6.

Figure 6

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Lucifer Yellow (LY) uptake by neonatal, juvenile and adult Langerhans' cells (LC). The in vitro uptake of LY via macropinocytosis at 37° and 4° was assessed in epidermal cells isolated from mice at 4 days and 6 weeks following birth (a). To account for any variations in non-specific or membrane receptor binding of the antigen, the mean fluorescence intensity at 37° was divided by the mean fluorescence intensity at 4°, generating a ratio for day 4 and adult LC at each time-point (b). To directly compare uptake between the various age-points, ratio data were then calculated for each age at each time-point (c). As shown in (c) LC from 4-day-old mice took longer to reach maximum uptake compared with adult LC (each point is representative of three separate experiments).

Discussion

Immune suppression resulting from cutaneous antigen treatment during the early neonatal period has been suggested to be, in part, the result of a decreased ability of epidermal LC to trap and internalize antigen.27 LC from 3-day-old epidermis lack expression of DEC-205 and demonstrate a reduction in MHC class II-stained dendritic processes.27 In this study we established that neonatal LC preferentially utilize a fluid-phase pathway to internalize both soluble and small particulate antigens, to compensate for the reduction in antigen uptake receptors.

As professional antigen-presenting cells, LC must be able to internalize mannose, as the majority of glycoproteins, such as those derived from bacteria and yeast, are mannosylated.29 To enhance the efficacy of this process, LC express members of the C-type lectin family of receptors, including DEC-20514,15 and Langerin.16 DEC-205 has been suggested to initiate uptake and strongly enhance human leucocyte antigen (HLA)-restricted presentation of mannose-conjugated antigens, and is up-regulated during maturation.14,30 An absence of lectin receptors may impact on the ability of LC to scavenge antigen. Our study has demonstrated that the expression of Langerin is greatly reduced on epidermal LC from 4-day-old mice, which is consistent with the previously established absence of DEC-205.27 A gradual acquisition of receptor expression occurs and, by day 14 following birth, the expression of Langerin is equivalent to that found within adult epidermis. These results indicate that LC in neonatal epidermis gradually mature during development.

The delay in surface expression of DEC-205 and Langerin on neonatal LC suggests that these cells have a reduced capacity to internalize antigen via a receptor-mediated pathway. This was reflected in the uptake of FITC–dextran at 37°, with adult LC internalizing more antigen than neonatal LC. However, adult LC demonstrated a higher fluorescence intensity at 4°. We postulate that this may be a result of the antigen adhering to the cell in a non-specific manner, or to cell-surface receptors. The latter hypothesis is feasible as adult LC express a greater number of receptors for mannose-type antigens than their neonatal counterparts. When ratio data were calculated to account for this increase in binding at 4°, it was demonstrated that, despite the lack of expression of DEC-205 and reduced expression of Langerin, LC from 4-day-old mice were more efficient at uptake than adult LC and, moreover, that they internalized antigen over a longer period of time before reaching maximum uptake. The reduction in FITC–dextran uptake by competition with unlabelled mannan and mannose was greater in adult LC than neonatal LC, suggesting that internalization of FITC–dextran by neonatal LC is less dependent on mannose-type receptors. An alternative and highly feasible explanation is that neonatal LC internalize small particulate antigens, such as FITC–dextran, via a receptor-independent pathway, such as fluid-phase macropinocytosis. Macropinocytosis would provide an efficient method of sampling the extracellular environment for soluble or non-binding antigens.25 Macropinocytosis utilizes the actin cytoskeleton to form macropinosomes, which are vesicles that arise when surface ruffles fold back against the cell wall or against each other.31 It has been previously demonstrated that 1 mg/ml of FITC–dextran is sufficient to saturate uptake via the mannose receptor; therefore differences in uptake between the age points will primarily reflect changes in macropinocytosis.29 As maximum FITC–dextran uptake occurs at a lower level in the adult than the neonate, this suggests that adult LC can be saturated at a lower concentration. This may be a result of the receptor level or, more likely, that neonatal LC can take up FITC–dextran via a pathway that is more difficult to saturate. As neonatal LC continued to internalize antigen over a longer period of time before reaching their maximum uptake, this is suggestive of a fluid-phase pathway. This hypothesis is supported by the efficient accumulation of the soluble antigen LY, previously shown to be a definitive marker of macropinocytosis.32 Together, these results strongly indicate that a fully functional macropinocytosis pathway is employed during the early neonatal period to internalize exogenous antigens. As previous studies by Sallusto et al. have demonstrated that uptake of LY is not inhibited by mannan,26 it is plausible that neonatal LC utilize fluid-phase uptake to internalize soluble and small particulate antigen to offset the reduction in receptor expression. It has been shown that in the presence of mannan, which is the functional equivalent of an absence of specific receptors, LC could still internalize mannosylated antigens in vitro in the fluid phase.15 We therefore suggest that macropinocytosis is up-regulated during the early neonatal period to ensure that the capacity to internalize antigen is not lost. This hypothesis is supported by the increase in inhibition of FITC–dextran accumulation in the presence of the specific phophatidylinositol 3-kinase inhibitor wortmannin, previously demonstrated to prevent closure of membrane ruffles, thereby reducing the pinocytic potential of a cell.33

The epidermal LC network undergoes significant cytoskeletal rearrangement during maturation.27 The decreased density of LC resident in 3-day-old epidermis, combined with the reduction in extensive dendrites, equates to an inability to form a semicontiguous network.27 As an adaptive mechanism, macropinocytosis may be up-regulated to ensure that the capacity to trap antigen is maintained. The lack of dendrites may also indirectly contribute to the increase in pinocytic activity demonstrated in vitro. As adult LC possess a well-developed dendritic network, little membrane is available for ruffling during macropinocytosis. Conversely, neonatal LC exhibit a more rounded morphology,27 therefore increasing available membrane. As a consequence, the magnitude of antigen internalized by macropinocytosis is increased. By day 14 following birth, the integrity of the LC network is improved,27 and it is at this stage of development that the amount of antigen internalized in vitro is reduced and parallel to adult controls. Together, these results suggest that the capacity to internalize antigen via a receptor-mediated pathway is not acquired until late in development. In contrast, the heightened in vitro fluid-phase activity of neonatal LC indicates that this pathway is functional from an early age. In vivo antigen exposure results in a reduction in the amount of antigen transported to the draining LN. Although ratio data suggests that, in vitro, neonatal LC are more efficient at antigen uptake than adult LC, antigen may still be transported to the LN via non-specific association of the antigen to the cell membrane or via non-metabolically active binding of antigen to surface receptors. It is also possible that although neonatal LC have an enhanced pinocytic activity, their ability to transport the internalized antigen to intracellular processing vesicles, and to load antigenic peptides onto newly synthesized MHC class II, is inept. The fate of ingested antigen, and thus the outcome of antigen trafficking to the draining LN, is worthy of future investigation.

We have previously demonstrated a gradual increase in the density of MHC class II+ cells in the epidermis during development, from relatively few LC in 3-day-old epidermis increasing to a level which, at day 14, is comparable to the number found in adult epidermis. As there is an influx of LC into the epidermis during the early neonatal period, yet these cells do not migrate following antigenic challenge, we propose that LC are actively retained in the epidermis to avoid inducing inappropriate immune responses at a time when the epidermal barrier is more susceptible to penetration. As a result of this decrease in cutaneous protection, one would expect an increase both of harmless soluble antigens as well as self- and protein particulate antigens within the epidermis. Rather than macropinosomes fusing with degradative lysosomes,34 neonatal LC may recycle antigen back to the extracellular space in a clearance and scavenging mechanism.35 In such a way, neonatal LC may use macropinocytosis to regulate the induction of immune responses to self-peptide.

Not all LC are retained in neonatal epidermis following antigenic treatment, as we have previously demonstrated a very low number of skin-derived LC resident in the node following cutaneous antigen treatment.27 We hypothesize that those few neonatal LC that are recruited to the LN do so in order to regulate immune responses to innocuous cutaneous antigen via a reduction in the total amount of antigen presented to naïve T cells. This hypothesis is supported by the recent findings of Geissman et al., who suggested that in chronically inflamed skin, LC migrate to the LN without associated maturation.36 Depending on further signals provided in the LN, these immature LC either induce tolerance or, following receipt of maturation stimuli, induce immunity. It is probable that during the neonatal period, the immature LC that do migrate to the draining LN induce tolerance, via a reduction in the two signals required for T-cell activation.

In conclusion, we have shown that the endocytic capacity of neonatal LC is unaffected by the absence of the C-type lectins DEC-205 and Langerin, and is acquired prior to their ability to initiate effective immunity. The aptitude of neonatal LC to sample their external environment without inducing immunity has important biological consequences for the protection against inappropriate responses during the developmental period.

Acknowledgments

The authors thank Mark Cozens for flow expertise, Dr Christina Trambas for review of the manuscript and the National Health and Medical Research Council and the Royal Hobart Hospital Research Foundation for their generous support.

Abbreviations

7-AAD

7-aminoactinomycin D

FCS

fetal calf serum

FITC

fluorescein isothiocyanate

HBSS

Hanks balanced salt solution

LC

Langerhans' cells

LN

lymph node

LY

Lucifer Yellow

MHC class II

major histocompatibility complex class II

PE

phycoerythrin

PBS

phosphate-buffered saline

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