Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 2001 May;103(1):61–69. doi: 10.1046/j.1365-2567.2001.01221.x

Acquisition of immune function during the development of the Langerhans cell network in neonatal mice

ANDREA L DEWAR 1, KATHLEEN V DOHERTY 1, GREGORY M WOODS 1, A BRUCE LYONS 1, H KONRAD MULLER 1
PMCID: PMC1783217  PMID: 11380693

Abstract

The immunological function of the Langerhans cell (LC) network in neonatal skin was examined by defining the development of cutaneous immunity relative to the structure, phenotype and function of the epidermal LC network in neonatal, juvenile and adult mice. Analysis of epidermal sheets showed the presence of major histocompatibility complex (MHC) II+, multilectin receptor DEC-205− cells within the epidermis of 3-day-old mice; both cell density and DEC-205 expression increased until day 14. When visualized with antibodies directed at MHC II, the network was poorly formed in 3- and 7-day-old mice, as there was a lower cell density and poor MHC II expression on dendritic processes, compared to mice at day14. Application of a fluorescent antigen to 3-day-old mice revealed that the LC were inefficient in transporting antigen to the draining lymph node. There was an improvement at day 7 and by day 14 comparable numbers of antigen carrying cells were detected in the lymph nodes of 6-week-old mice. The reduced antigen carriage in 3- and 7-day-old mice correlated with a poor contact sensitivity response. This was not simply due to failure to present antigen, but development of immunosuppression, as transfer of T cells from adult mice that were previously treated with antigen when they were 3 days old, to adult recipients resulted in antigen specific immunosuppression. Analysis of CD80 and CD86 expression showed that LC from day 3 skin expressed CD80, but not CD86 and application of antigen through this skin was inefficient in upregulating CD86. These findings indicate that when the neonatal LC network is poorly developed it is functionally immature and antigen applied through this ‘functionally immature network’ results in antigen specific immunosuppression.

Introduction

Langerhans cells (LC) are intraepidermal dendritic cells (DC) which form an extensive interlinking network throughout the epidermis to trap cutaneous antigen1. Following uptake of antigen, LC migrate via the afferent lymphatics to the lymph nodes2 and during transit they undergo differentiation from an antigen processing to an antigen-presenting phenotype3. The antigen-processing phenotype is characterized by the expression of major histocompatibility complex (MHC) II and the multilectin receptor DEC-205, while maturation to the presenting phenotype leads to upregulation of MHC II and costimulatory molecules4. Once in the lymph nodes, presentation of antigen to T cells results in the induction of an immune response specific for the antigen encountered in the skin5.

We have previously demonstrated that the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) depletes the epidermis of LC6 and that application of a contact sensitizer through DMBA-treated skin induces antigen-specific immunosuppression7. This led to the conclusion that, while an extensive epidermal LC network is crucial for the induction of a positive immune response, an incompletely formed LC network may specifically result in suppressive immune responses.

The density of LC in the epidermis is not the only important factor in effective cutaneous immunity, as the interaction between the T cell and antigen-presenting cell within the lymph node also determines the outcome of the immune response. Costimulatory molecules, including CD40,8,9 CD80 and CD86,10 adhesion molecules11 and soluble mediators such as interleukin (IL)-1012 and IL-1213 influence the immune response. In the absence of adequate costimulation,14 or in the presence of cytokines such as IL-10,15 the immune response is directed towards a tolerogenic or a T helper 2 pathway.16,17

The differentiation of LC from an antigen uptake and processing phenotype to potent antigen-presenting function has been well defined in recent years.5,18 However, very little is known about the events that lead to the LC network itself becoming functionally competent in the neonate. While LC have been identified in fetal skin by either MHC class II expression or ATPase staining in humans19 and mice,20 little is known of their functional maturity or potential to drive immune responses. Maturation of LC occurs during the early postnatal period and cells with dendritic morphology are evident in the skin by day 19 of gestation,21 however, Birbeck granules are not detectable until day 4 postpartum.22 This raises the question of the functional competence of neonatal LC, particularly, prior to day 4. During this neonatal period exposure of the immune system leads to tolerance rather than immunity. Deviation of the immune response toward a T helper type 2 response has been proposed to account for this23 and evidence is accumulating that dendritic cells play a key role in driving this outcome.24

We propose that an important correlation exists between the maturity of the LC network and development of immune function in the neonate. In particular, the hypothesis that application of antigen to an immature LC network results in immunosuppression was assessed as this may have a crucial role in neonatal tolerance. In order to evaluate this, the structure, phenotype and function of the epidermal LC network of neonatal, juvenile and adult mice was compared. This was then correlated to the acquisition of immune function.

Materials and methods

Animals

Neonatal (3 days old), juvenile (7 and 14 days old) and adult (6–12 weeks old, male) BALB/c (H-2d) mice were obtained from the University of Tasmania Central Animal House. Mice were provided with food and water ad libitum and used with the permission of the University of Tasmania Ethics Committee (Animal Experimentation; permit number A5519).

Epidermal sheets

Neonatal, juvenile and adult mice were killed by CO2 asphyxiation, and epidermal sheets were prepared from the dorsal skin using a modified ethylenediamine tetra-acetic acid (EDTA) separation procedure25 first described by Baker and Habowsky.26 Briefly the dorsal hair was removed using animal clippers and/or depilatory cream (Veet; Reckitt and Colman, Sydney, Australia). Skin was washed thoroughly with tap water and the keratin layer removed by stripping with sticky tape. Fresh sticky tape was applied to the hair-free region of skin and the sticky tape–skin combination excised. Skin segments were incubated in 20 mm EDTA/phosphate-buffered saline (PBS) for 2 hr at 37° with agitation, after which the dermis was mechanically separated from the epidermis.

The prepared sheets were fixed in acetone for 10 min and then incubated in a culture supernatant containing anti-MHC-II monoclonal antibody (TIB120) or anti-DEC205 (NLDC-145)27, for 2 hr at 37°, followed by overnight at 4°. Epidermal sheets were washed three times with PBS prior to incubation in a secondary antibody–peroxidase conjugate for 2 hr at 37° (goat anti-rat immunoglobulin G (IgG) horseradish peroxidase conjugate; Southern Biotechnology Associates Ltd, Birmingham, AL). After three washes with PBS, sheets were incubated in diaminobenzidine (Sigma, St Louis, MO) at room temperature until a brown colour change was observed. Sheets were washed three times with PBS, mounted in phosphate-buffered glycerol jelly and stored at 4°.

Epidermal sheets were examined using the public domain NIH Image program (written by Wayne Rasband at the US.National Institutes of Health (Bethesda, MD) and available on the Internet by anonymous ftp from zippy.nimh.nih.gov). Five fields of view were captured per antibody-stained sheet per mouse using a black and white Galai CCD video camera, with a minimum of six mice examined per age group. The density of NLDC-145 (i.e DEC205) and TIB-120 (i.e. MHC-II) cells was calculated for each captured image.

Epidermal cell suspensions

Adult and neonatal mice were killed and the dorsal and ventral hair removed by means of animal clippers and/or depilatory cream (Veet: Reckitt and Colman). The skin was washed thoroughly with lukewarm tap water and excised. Excised skin was cut into small squares and incubated in Hanks' balanced salt solution (HBSS; Gibco BRL Life Technologies, Melbourne, Australia)/0·5% dispase (Gibco BRL Life Technologies)/100 U/ml DNase (Amersham Life Sciences, Sydney, Australia) at 37° for 2 hr. The epidermis was mechanically separated from the dermis by gentle scraping with blunt-nosed forceps, or in the case of neonatal mice, peeled off using curved, fine forceps. The resultant epidermis was incubated in fresh HBSS/0·5% dispase/100 U/ml DNase at 37° for 20 min, after which time an equal volume of complete Dulbecco's modified Eagle's minimal essential medium (DMEM; ICN Biomedicals Ltd, Sydney, Australia) was added. The solution was swirled for 5 min and filtered through cotton wool to remove large particles and the resultant epidermal cell suspension was washed three times with DMEM supplemented with 10% fetal calf serum (FCS; CSL Biosciences, Melbourne, Australia) and the final pellet resuspended in DMEM−10% FCS. Cell suspensions were enriched for LC by underlaying 5 ml of the epidermal cell suspension with 2 ml of 18% metrizamide (Nycomed, Oslo, Norway; 18 g plus 100 ml medium DMEM supplemented with 10% fetal calf serum) and centrifuged for 10 min at 600 g. Langerhans cells at the interface were collected and washed twice with DMEM−10% FCS prior to staining.

Flow cytometry

Enriched epidermal cell suspensions were initially blocked with 5% normal mouse serum at 4° for 30 min, excluding those cells stained for Fc receptors. Following blocking, approximately 5 × 105 epidermal cells were incubated at 4° for 30 min with either biotinylated anti-mouse CD80 (Clone 1610A1, Cat. no. 09602D, PharMingen, San Diego, CA) or biotinylated anti-mouse CD86 (Clone GL1, Cat. no. 09272D PharMingen). After washing, streptavidin-allophycocyanin (Amersham) was added at 4° for 30 min, and washed a further three times with PBS/2% bovine serum albumin (BSA)/0·1% sodium azide. The fluorescence intensity of stained epidermal cell suspensions was examined using a Coulter ELITE ESP flow cytometer, and analysis performed using CellQuest® version 3.1f software.

Contact sensitivity response

Sensitization.

Neonatal (3 days), juvenile (7 and 14 days) and adult mice were treated on the shaved dorsal trunk with 10 µl, 20 µl, 50 µl and 100 µl, respectively, of 2% trinitrochlorobenzene (TNCB) (TCI, Tokyo) or the acetone vehicle alone. These volumes were selected as they represented the same relative surface area for each mouse at the different age groups.

Resensitization.

Six weeks later, a period required for the neonatal mice to mature, 100 µl 2% TNCB, or the vehicle alone, was reapplied to the shaved dorsal skin.

Challenge.

The right ears of both treated and control mice were challenged 5 days later with 20 µl 0·5% TNCB, and the left ears with 20 µl of the vehicle solution. The thickness of the right ear (challenged) and the left ear (control) was measured 48 hr after treatment using an engineer's spring-loaded micrometer. The percentage increase in ear thickness was calculated using the following formula: [(thickness challenged ear − thickness control ear)/thickness control ear] × 100

Analysis of antigen carriage to lymph nodes

Neonatal (3 days), juvenile (7 and 14 days) and adult mice were treated with 20, 40, 100, 200, respectively, of fluorescein isothiocyanate (FITC; Sigma) 0·5% dissolved in 1:1 acetone:dibutylphthalate) and the brachial, axillary and inguinal lymph nodes removed 20 hr later. Lymph node cell suspensions were layered onto 2 ml metrizamide (Nycomed; 14·5 g plus 100 ml medium RPMI-1640 supplemented with 10% fetal calf serum) and centrifuged for 10 min at 600 g. Dendritic cells at the interface were collected, washed once and resuspended in RPMI-1640–10% FCS. The total dendritic cell (DC) population was identified by flow cytometric analysis gating for the cells with high forward- and side-scatter properties as this was primarily the DC population. These cells were examined for fluorescence using a Coulter ELITE ESP flow cytometer and CellQuest® version 3.1f software.

Adoptive transfer of spleen cells

Spleens were removed from adult male mice 5 days after they were sensitized with 2% TNCB at 3 days or 6 weeks of age followed 6 weeks later by a further 2% TNCB. Single-cell suspensions were prepared in PBS/0·1% BSA and washed three times. Cell viability calculated by trypan blue exclusion. Recipient mice were injected with 5 × 107 viable lymphocytes via the lateral tail vein. Control mice received spleen cells from acetone treated donors or received no spleen cells at all. Within 2 hr of injection, recipient mice were treated with 100 µl 2% TNCB or 200 µl 0·5% DNFB (Sigma) to the shaved dorsal skin. Mice were challenged with 20 µl 0·5% TNCB or 0·2% 2,4-dinitrofluorobenzene (DNFB) on the right ear and 20 µl vehicle solution on the left ear 5 days later. Ear thicknesses were measured 48 hr later using an engineer's spring-loaded micrometer.

Statistical analysis

To determine whether differences between groups were statistically significant, the data was examined using a Student's unpaired, two-tailed, t-test. Data sets were considered to be significantly different when the probability value < 0·05.

Results

Evaluation of the Langerhans cell network

Epidermal sheets from neonatal, juvenile and adult mice were stained for MHC II, a molecule that typically identifies LC within the epidermis, and DEC-205, a putative antigen-uptake receptor expressed on mature LC. At all ages, MHC II+ cells were identifiable within the epidermis, however, a significant difference in the expression of DEC-205 was observed (Fig. 1). While LC within neonatal epidermis expressed MHC II, DEC-205 expression was absent. DEC-205 expression substantially increased by day 7, but was evident on significantly fewer cells than MHC II. By 14 days of age MHC II and DEC-205 expression was equivalent with adult epidermis.

Figure 1.

Figure 1

Open in a new tab

Expression of MHC II and DEC-205 on neonatal, juvenile and adult LC. Epidermal sheets prepared from mice at 3 days (top panel), 7 days (2nd from top panel), 14 days (2nd from bottom panel) and 6 weeks (bottom panel), were acetone fixed and stained for MHC II (left panel) and DEC-205 (right panel). Staining was visualized with DAB. Note the absence of DEC-205 staining in epidermal sheets from 3-day-old mice, the round morphology of cells at 3 and 7 days and the dendritic morphology at 6 weeks.

Morphological analysis of epidermal sheets demonstrated that the LC networks in 3-day-old (neonatal) and 7-day-old (juvenile) mice were poorly established (Fig. 1). Langerhans cells in the epidermis of neonates were morphologically distinct when visualized with MHC II. They were characterized by large, rounded cell bodies and limited visible dendrites. As the mice matured the dendritic processes became more evident and interconnection of MHC-II+ dendritic processes between adjacent cells was evident.

In order to clarify the relationship between DEC-205 and MHC II expression, the density of cells expressing each of these markers was quantified. As shown in Fig. 2, the density of MHC II+ cells in 3-day-old neonatal mice, was approximately 1200 cells/mm2, which increased to approximately 1800 cells/mm2 by day 14. Interestingly, the density of MHC II+ cells in adult epidermis was significantly lower than that observed in 14-day-old mice.

Figure 2.

Figure 2

Open in a new tab

Density of MHC II+ and DEC-205+ cells within epidermis of neonatal, juvenile and adult mice. Epidermal sheets prepared from mice at 3, 7 and 14 days, and 6 weeks were acetone fixed and stained with anti-MHC II (filled bars) and anti-DEC-205 (open bars) antibodies. Staining was visualized with diaminobenzidine (DAB). Cells were enumerated and density calculated using NIH Image software. Results shown are mean LC density (± SEM) from five fields obtained from six separate animals.

Langerhans cells expressing DEC-205 cells were not detected until 7 days of age and the density peaked in 14-day-old mice; this was greater than adult mice. The difference in expression of MHC II and DEC-205 was most marked in 3-day-old neonatal mice where the density of MHC II+ cells was equivalent to that observed in adults, but DEC-205 expression was absent. Although LC from 7-day-old mice expressed DEC-205, the density of cells positive for this molecule was still significantly lower than the density of cells expressing MHC II. By 14 days of age the density of cells expressing MHC II was equivalent to the cells expressing DEC-205.

Detection of FITC+ cells in draining lymph nodes

The phenotypic and structural immaturity of the LC network in neonatal and 7-day-old mice suggested that these cells might be functionally deficient. One of the key functions of LC is to transport antigen from the skin to the lymph nodes. To examine the ability of LC to take up and transport antigen, FITC was applied to the dorsal skin of neonatal, juvenile and adult mice. Twenty-four hours later the lymph nodes were removed and analysed for the presence of FITC+ cells.

As shown in Fig. 3, a low level of FITC was detected in a small number of cells in the draining lymph nodes of mice treated at 3 days of age. Similarly, a single population of cells carrying a low level of FITC was detected in mice treated at 7 days of age. As a large number of cells carried this low level of FITC, the total amount of FITC arriving in the lymph nodes of mice treated at 7 days was higher than that seen in mice treated at 3 days.

Figure 3.

Figure 3

Open in a new tab

Antigen transport by neonatal, juvenile and adult LC. FITC was applied to the dorsal skin of mice at 3, 7 and 14 days, and 6 weeks. Control mice were treated with acetone alone. 20 hr later draining lymph nodes were removed and dendritic cells were enriched on a 14·5% metrizamide gradient. The solid histogram shows the profile of dendritic cell associated fluorescence indicative of FITC uptake and transport to the node. The dotted histogram is fluorescence intensity of DC from control animals.

Analysis of FITC carriage by cells from mice treated at 14 days of age demonstrated that two populations of cells existed on the basis of mean fluorescence intensity. This included a population of cells carrying a small amount of FITC (FITClo) and a larger population of cells carrying significantly more FITC (FITChi). The total number of cells carrying FITC was similar to that observed in mice treated at 7 days of age. FITChi and FITClo populations were also seen in mice treated as adults. The fluorescence intensity associated with each of these populations was 10 times greater than that seen in mice treated at 14 days of age.

Contact sensitivity response to TNCB

To determine whether the maturity of the LC network correlated with the ability to initiate an immune response, the contact sensitivity response of mice sensitized initially at 3, 7, 14 days and 6 weeks of age was examined 6 weeks after sensitization. A comparison of the contact sensitivity response elicited in these mice revealed that the magnitude of the response was directly related to the age of the animal at sensitization (Fig. 4). While the immune response of mice sensitized at 14 days of age was not significantly different to that observed in mice sensitized as adults with both showing a 50% increase in ear thickness, mice sensitized initially as neonates or at 7 days of age gave significantly lower contact sensitivity responses. This decreased ability to respond immunologically to antigen was most notable in mice sensitized at 3 days where the response was 65% less than that seen in mice sensitized as adults. Mice sensitized as adults were immunologically ‘responsive’, while mice sensitized as neonates gave ‘suppressed’ immune responses.

Figure 4.

Figure 4

Open in a new tab

Contact sensitivity response to TNCB is dependent on the age of mice at priming. Mice were sensitized (sens.) by application of TNCB to dorsal skin at 3 days, 7 days, 14 days or 6 weeks of age. Six weeks later mice were resensitized (resens.) and the contact sensitivity response was measured following ear challenge. Results show the mean percentage increase in ear thickness (± SEM). Mice sensitized at 3 and 7 days displayed a significantly reduced response to challenge when compared with mice sensitized at 6 weeks (P = < 0·05; NS = not significant; Student's unpaired t-test).

Mechanism and specificity of immune suppression

Adoptive transfer experiments were performed to determine whether the response observed in immune suppressed mice was cell mediated. Spleen cells were transferred from naive, responsive or suppressed donors to adult recipients, which were subsequently challenged with TNCB. Results illustrated in Fig. 5 show that the transfer of spleen cells from responsive donors to naive recipients resulted in a contact sensitivity response, which was similar to that in the responsive donor mice. This response was equivalent to that seen when naive spleen cells were transferred. In contrast, the transfer of spleen cells from suppressed donors resulted in a significantly reduced contact sensitivity response in recipient mice.

Figure 5.

Figure 5

Open in a new tab

The suppressed contact sensitivity response is transferable and antigen specific. Mice were sensitized (sens.) by application of TNCB to dorsal skin at 3 days or 6 weeks of age and 6 weeks later mice were resensitized (resens.). Spleen cells were transferred from these mice to naïve adult recipients. These recipients were then sensitized with TNCB (filled bars) or DNFB (open bars) and the contact sensitivity response following ear challenge was assessed. Results shown are the mean percentage increase in ear thickness in groups of 6 mice (± SEM). The response to TNCB is significantly suppressed (P < 0·01) in recipient mice that received spleen cells from donor mice initially treated at 3 days with TNCB while the response to DNFB was not suppressed. (NS = not significant).

To determine whether the transferred immune suppression was antigen specific, mice receiving spleen cells from responsive or suppressed donors were sensitized and challenged with a different contact sensitizer, DNFB. The transfer of spleen cells from responsive, naive or suppressed donors did not significantly affect the contact sensitivity response of recipient mice to DNFB (Fig. 5) indicating that an antigen-specific suppressive mechanism was involved.

Analysis of costimulatory molecule expression

It was apparent that at 3 days the epidermal LC were deficient in their ability to stimulate an immune response. This deficiency was further explored by evaluating the expression of CD80 and CD86. Figure 6 shows that CD86 was not expressed on LC from the epidermis of 3-day-old mice in contrast to CD80 which showed weak expression. Further analysis of CD86 expression demonstrated that following exposure to antigen, CD86 expression was upregulated on LC from the skin of 3-day-old mice, but this level of expression was still lower than the LC from adult mice.

Figure 6.

Figure 6

Open in a new tab

(a) Surface expression of CD80 and CD86 on epidermal LC. Epidermal cells were isolated from skin and the preparation was enriched for LC by metrizamide gradient centrifugation. Expression of CD80, but not CD86, was apparent on the LC of mice at day 3. (b) Further analysis of CD86 expression revealed that when FITC was applied to the skin of 3-day-old mice the FITC+ve LC in the draining lymph nodes expressed less CD86 compared to LC from adult mice. The profiles are representative of three reproducible experiments. The dotted histogram is the isotype control.

Discussion

A comprehensive epidermal LC network is essential for the induction of an immune response to antigen encountered in the skin.28 This study concentrated on the in vivo development of the LC network in neonatal mice in order to determine the relationship between the developmental status of the LC network and the capacity to initiate an immune response to cutaneous antigen.

We established that the LC network undergoes significant change as the animal matures. Despite the observation of MHC-II+ cells, indicative of LC, in the epidermis of mice at day 3 and at the same density as adult mice, the cellular distribution of MHC-II expression was restricted to the cell body. The absence of MHC-II+-stained dendritic processes gave the impression of a poor cellular network. This was partially compensated by an increase in the density of MHC II+ cells in the 7-day-old mice. As the animals matured, the decrease in the density of MHC-II+ cells was accompanied by an increase in MHC II expression on dendritic processes leading to an improvement in the integrity of the LC network. Cells with dendritic morphology have previously been identified in fetal epidermis by staining for ADPase21 suggesting that dendritic processes are present on LC within the skin of neonates, but it remains plausible that the dendritic cell processes are deficient in MHC II expression. To maintain its functional integrity as the animal grows, the LC network, by extending dendritic contact between neighbouring LC, adapts to the reduced cell density. Analysis of MHC II expression on the dendrites is an indicator of this development and suggests that expression of MHC II on dendritic processes is a marker for functional maturity. Gradual acquisition of MHC-II on cells within the epidermis of BALB/c mice has been reported previously, with class II being detected at day 19 of gestation on a limited number of cells.20

The increase in the density of MHC-II+ cells has been ascribed to upregulation of MHC-II on resident MHC-II− cells within the epidermis.21 We have confirmed that the expression of class II increases as the skin develops and that the distribution of class II on the cells also changes. Our data also suggests that acquisition of DEC-205 occurs subsequent to MHC class II acquisition since the density of cells expressing DEC-205 in 7-day-old mice was significantly lower than the density of cells expressing MHC-I. It was not until the LC network approached morphological maturity in 14-day-old mice that the density of cells expressing DEC-205 and MHC II equilibrated. DEC-205 has been identified as a putative antigen-uptake receptor, which is upregulated during maturation,29 suggesting that the absence of DEC-205 expression by LC in neonates may impact on the ability of these cells to scavenge antigen. Assessment of in vivo antigen carriage supports this hypothesis as the intensity of FITC staining of lymph node cells correlated with the development of the LC network and with DEC-205 expression.

The absence of cells bearing high levels of fluorescent antigen in neonatal animals supports the hypothesis that neonatal LC are functionally deficient in antigen uptake. Other studies utilizing fluorescent haptens have concluded that the appearance of fluorescent cells within the node following topical application is the consequence of active uptake and migration of epidermal LC.2,30 However, it has recently been reported that haptens applied topically may also enter the node via local lymphatics independently of cellular association.31 Given that the concentration of hapten used in this study was significantly lower than that used by Pior et al.31 and that the intensity of cell associated fluorescence is dependent on the concentration, rather than volume of hapten used (data not shown), the difference in fluorescence intensity observed between neonatal and juvenile mice is significant and supports our interpretation of a difference in the functional capacity of the cells to take up antigen. Nonetheless, a contribution to the low level of FITC fluorescence in the draining lymph nodes could also be due to the dispersion of free FITC via the afferent lymphatics which could be trapped by lymph node dendritic cells. Such an outcome will also occur if neonatal LC are deficient in antigen uptake and subsequent migration, as low levels of FITC would reach the draining lymph nodes.

In addition to the requirement for the expression of molecules involved in antigen uptake and presentation by LC, the costimulatory molecules CD80 and CD86 are required for effective T-cell activation.32 Striking differences were observed in the expression of the costimulatory molecule CD86 by LC, as epidermal cells isolated from 3-day-old neonatal mice failed to express this molecule, in contrast to epidermal cells from juvenile and adult mice. This correlated directly to the time period where immunosuppression was evident.

A key feature of the chemical carcinogen model of immunosuppression is the low antigen load associated with LC derived from carcinogen-treated skin which correlates directly with induction of immunosuppression.33 Therefore, the potential of neonatal cells to initiate contact sensitivity responses was of special interest, as they bear some of the hallmarks of LC found in the chemical carcinogen model of immunosuppression. The immature nature of the LC network in neonatal mice, reduced CD86 expression, the absence of DEC-205 expression and the consequent decrease in their capacity to transport antigen to the lymph nodes suggests that these cells would be less effective at initiating an immune response to cutaneous antigen. This prediction was supported by contact sensitivity experiments, which demonstrated that when mice were originally sensitized as neonates, their immune response was significantly impaired when compared to the adult response. The magnitude of the response related directly to the integrity and phenotype of the LC network at the time of sensitization. Thus, the poor response in mice sensitized when the LC network was inadequately formed, was caused by limited antigen uptake, low levels of antigen carriage and migration of few cells to the draining lymph node. The contact sensitivity response improved as the LC network developed in concert with increased antigen carriage. Hence, sensitization of mice at 14 days of age resulted in a response similar to the adult, owing to the presence of a structurally and phenotypically mature LC network, which was effective at both scavenging and transporting antigen. However the 10 fold difference in antigen carriage between day 14 and adult LC suggests that full functional potential had not yet been attained by day 14. This correlates with the delay in acquisition of immunocompetence seen within the respiratory tract that has been attributed to the limited maturation of dendritic cells within airway epithelium.34

That antigen presentation occurred in the neonates despite the low contact sensitivity response and limited migration of antigen-carrying cells, is supported by the adoptive transfer experiments, which demonstrate transfer of antigen-specific immune suppression. In order for cell-mediated antigen specific immune suppression to occur, antigen must be presented to the appropriate regulatory T-cell population. It is evident that the induction of regulatory cells35 is not only a consequence of the maturation status of the antigen-presenting cell, but the level of antigen carried by these cells as well as a reduced expression of CD86.

This study supports the hypothesis that neonatal LC may specifically initiate suppressive rather than active immune responses. The phenomena of neonatal tolerance and the patterns of T-cell reactivity against the antigens encountered during the neonatal period that determine the outcome of the response in adulthood have been well described.36 Mechanisms included deletion of antigen-specific T cells37 and induction of regulatory T cells.23 Antigen-specific tolerance has been ascribed to the inherent properties of neonatal T cells, including defective IL-2 production, and skewing of the immune response toward a T helper type 2 response.23 Other studies suggest that neonatal T cells can be primed for both T helper type 1 and type 2 responses,24,38 but the outcome depends mainly on the mode of immunization.23 That the nature of the antigen-presenting cell determines whether the outcome is effective priming of immunity, or tolerance, supports the hypothesis that the antigen-presenting cell is central to induction of tolerance versus immunity in the neonate.24

The concept of the antigen-presenting cell dictating the outcome of the immune response extends beyond the neonatal period. LC mediated activation of antigen-specific immunosuppression also occurs when antigen is applied to adult mouse skin, which has been depleted of LC by prior exposure to the chemical carcinogen, DMBA7,39 or to skin previously exposed to ultraviolet light (UVB).40 Both of these agents alter the epidermal LC network resulting in reduced LC numbers and changes in dendritic morphology. The ability of LC to initiate an immune response is also dependent on the presence of a structurally mature network of LC. For example, application of antigen to benzo-(a)-pyrene-treated skin resulted in a significant reduction in the contact sensitivity response despite the skin containing a high density of LC; however, the LC had shortened dendritic processes and a poorly formed network.41 This situation is analogous to the increased LC number and reduced dendrite development observed with epidermal LC in neonatal mice described in this study.

Finally, control of neonatal tolerance does not reside exclusively within the T-cell population as neonatal T cells can be activated efficiently by adult LC.24 Our study supports the concept that LC have a central role in determining whether tolerance or active immunity to cutaneous antigen occurs in the neonate. While the LC network is immature, tolerance is the inevitable outcome caused by the inability of the neonatal LC to stimulate effective immunity.

Acknowledgments

The authors thank the National Health and Medical Research Council, the Cancer Council of Tasmania and the Royal Hobart Hospital Research Foundation for their generous support.

References

  • 1.Shelley JB, Juhlin L. Langerhans cells form a reticuloepithelial trap for external contact sensitizers. Nature. 1976;261:46. doi: 10.1038/261046a0. [DOI] [PubMed] [Google Scholar]
  • 2.Kripke ML, Munn CG, Jeevan A, Tang J, Bucana C. Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization. J Immunol. 1990;145:2833–8. [PubMed] [Google Scholar]
  • 3.Larsen CP, Steinman RM, Witmer-Pack M, Hankins DF, Morris PJ, Austyn JM. Migration and maturation of Langerhans cells in skin transplants and explants. J Exp Med. 1990;172:1483–93. doi: 10.1084/jem.172.5.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hart DN, Clark GJ, Dekker JW, et al. Dendritic cell surface molecules. A proliferating field. Adv Exp Med Biol. 1997;417:439–42. doi: 10.1007/978-1-4757-9966-8_72. [DOI] [PubMed] [Google Scholar]
  • 5.Hart DN. Dendritic cells unique leukocyte populations which control the primary immune response. Blood. 1997;90:3245–87. [PubMed] [Google Scholar]
  • 6.Muller HK, Halliday GM, Knight BA. Carcinogen-induced depletion of cutaneous Langerhans cells. Br J Cancer. 1985;52:81–5. doi: 10.1038/bjc.1985.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Halliday GM, Muller HK. Induction of tolerance via skin depleted of Langerhans cells by a chemical carcinogen. Cell Immunol. 1986;99:220–7. doi: 10.1016/0008-8749(86)90230-3. [DOI] [PubMed] [Google Scholar]
  • 8.Peguet-Navarro J, Dalbiez-Gauthier C, Rattis FM, Van Kooten C, Banchereau J, Schmitt D. Functional expression of CD40 antigen on human epidermal Langerhans cells. J Immunol. 1995;155:4241–7. [PubMed] [Google Scholar]
  • 9.Cella M, Scheidegger D, Palmer Lehmann K, Lane P, Lanzavecchia A, Alber G. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity. T–T help via APC activation. J Exp Med. 1996;184:747–52. doi: 10.1084/jem.184.2.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Larsen CP, Ritchie SC, Pearson TC, Linsley PS, Lowry RP. Functional expression of the costimulatory molecule, B7/BB1, on murine dendritic cell populations. J Exp Med. 1992;176:1215–20. doi: 10.1084/jem.176.4.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chang CH, Furue M, Tamaki K. Selective regulation of ICAM-1 and major histocompatibility complex class I and II molecule expression on epidermal Langerhans cells by some of the cytokines released by keratinocytes and T cells. Eur J Immunol. 1994;24:2889–95. doi: 10.1002/eji.1830241146. [DOI] [PubMed] [Google Scholar]
  • 12.Enk AH, Angeloni VL, Udey MC, Katz SI. An. essential role for Langerhans cell-derived IL-1 beta in the initiation of primary immune responses in skin. J Immunol. 1993;150:3698–704. [PubMed] [Google Scholar]
  • 13.Heufler C, Koch F, Stanzl U, et al. Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells. Eur J Immunol. 1996;26:659–68. doi: 10.1002/eji.1830260323. [DOI] [PubMed] [Google Scholar]
  • 14.Harding FA, Allison JP. CD28–B7 interactions allow the induction of CD8+ cytotoxic T lymphocytes in the absence of exogenous help. J Exp Med. 1993;177:1791–6. doi: 10.1084/jem.177.6.1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Chang CH, Furue M, Tamaki K. B7-1 expression of Langerhans cells is up-regulated by proinflammatory cytokines, and is down-regulated by interferon-gamma or by interleukin-10. Eur J Immunol. 1995;25:394–8. doi: 10.1002/eji.1830250213. [DOI] [PubMed] [Google Scholar]
  • 16.Steinbrink K, Wolfl M, Jonuleit H, Knop J, Enk AH. Induction of tolerance by IL-10-treated dendritic cells. J Immunol. 1997;159:4772–80. [PubMed] [Google Scholar]
  • 17.Cua DJ, Coffman RL, Stohlman SA. Exposure to T helper 2 cytokines in vivo before encounter with antigen selects for T helper subsets via alterations in antigen-presenting cell function. J Immunol. 1996;157:2830–6. [PubMed] [Google Scholar]
  • 18.Cohen PJ, Katz SI .Cultured human Langerhans cells process and present intact protein antigens. J Invest Dermatol. 1992;99:331–6. doi: 10.1111/1523-1747.ep12616663. [DOI] [PubMed] [Google Scholar]
  • 19.Foster CA, Holbrook KA, Farr AG. Ontogeny of Langerhans cells in human embryonic and fetal skin. expression of HLA-DR and OKT-6 determinants. J Invest Dermatol. 1986;86:240–3. doi: 10.1111/1523-1747.ep12285201. [DOI] [PubMed] [Google Scholar]
  • 20.Romani N, Schuler G, Fritsch P. Ontogeny of Ia-positive and Thy-1-positive leukocytes of murine epidermis. J Invest Dermatol. 1986;86:129–33. doi: 10.1111/1523-1747.ep12284135. [DOI] [PubMed] [Google Scholar]
  • 21.Elbe A, Tschachler E, Steiner G, Binder A, Wolff K, Stingl G. Maturational steps of bone marrow-derived dendritic murine epidermal cells. Phenotypic and functional studies on Langerhans cells and Thy-1+ dendritic epidermal cells in the perinatal period. J Immunol. 1989;143:2431–8. [PubMed] [Google Scholar]
  • 22.Kobayashi M, Asano H, Fujita Y, Hoshino T. Development of ATPase-positive, immature Langerhans cells in the fetal mouse epidermis and their maturation during the early postnatal period. Cell Tissue Res. 1987;248:315–22. doi: 10.1007/BF00218198. [DOI] [PubMed] [Google Scholar]
  • 23.Forsthuber T, Yip HC, Lehmann PV. Induction of TH1 and TH2 immunity in neonatal mice. Science. 1996;271:1728–30. doi: 10.1126/science.271.5256.1728. [DOI] [PubMed] [Google Scholar]
  • 24.Ridge JP, Fuchs EJ, Matzinger P. Neonatal tolerance revisited: turning on newborn T cells with dendritic cells [see comments] Science. 1996;271:1723–6. doi: 10.1126/science.271.5256.1723. [DOI] [PubMed] [Google Scholar]
  • 25.Gabrilovich DI, Woods GM, Patterson S, Harvey JJ, Knight SC. Retrovirus-induced immunosuppression via blocking of dendritic cell migration and down regulation of adhesion molecules. Immunology. 1994;82:82–7. [PMC free article] [PubMed] [Google Scholar]
  • 26.Baker KW, Habowsky JEJ. EDTA separation and ATPase Langerhans cell staining in the mouse epidermis. J Invest Dermatol. 1983;80:104–7. doi: 10.1111/1523-1747.ep12531712. [DOI] [PubMed] [Google Scholar]
  • 27.Kraal G, Breel M, Janse M, Bruin G. Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody. J Exp Med. 1986;163:981–97. doi: 10.1084/jem.163.4.981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Toews GB, Bergstresser PR, Streilein JW, Sullivan S. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin-painting with DNFB. J Immunol. 1980;124:445–53. [PubMed] [Google Scholar]
  • 29.Kato M, Neil TK, Clark GJ, Morris CM, Sorg RV, Hart DNJ. cDNA cloning of human DEC-205, a putative antigen-uptake receptor on dendritic cells. Immunogenetics. 1998;47:442–50. doi: 10.1007/s002510050381. 10.1007/s002510050381. [DOI] [PubMed] [Google Scholar]
  • 30.Macatonia SE, Knight SC, Edwards AJ, Griffiths S, Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. J Exp Med. 1987;166:1654–67. doi: 10.1084/jem.166.6.1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Pior J, Vogl T, Sorg C, Macher E. Free hapten molecules are dispersed by way of the bloodstream during contact sensitization to fluorescein isothiocyanate. J Invest Dermatol. 1999;113:888–93. doi: 10.1046/j.1523-1747.1999.00770.x. 10.1046/j.1523-1747.1999.00770.x. [DOI] [PubMed] [Google Scholar]
  • 32.Jenkins MK, Johnson JG. Molecules involved in T-cell costimulation. Curr Opin Immunol. 1993;5:361–7. doi: 10.1016/0952-7915(93)90054-v. [DOI] [PubMed] [Google Scholar]
  • 33.Woods GM, Doherty KV, Malley RC, Rist MJ, Muller HK. Carcinogen-modified dendritic cells induce immunosuppression by incomplete T-cell activation resulting from impaired antigen uptake and reduced CD86 expression. Immunology. 2000;99:16–22. doi: 10.1046/j.1365-2567.2000.00928.x. 10.1046/j.1365-2567.2000.00928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nelson DJ, McMenamin C, McWilliam AS, Brenan M, Holt PG. Development of the airway intraepithelial dendritic cell network in the rat from class II, major histocompatibility (Ia)-negative precursors. differential regulation of Ia expression at different levels of the respiratory tract. J Exp Med. 1994;179:203–12. doi: 10.1084/jem.179.1.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Asherson GL, Zembala M, Thomas WR, Perera MACC. Suppressor cells and the handling of antigen. Immunol Rev. 1980;50:3. doi: 10.1111/j.1600-065x.1980.tb00306.x. [DOI] [PubMed] [Google Scholar]
  • 36.Holt PG, Macaubas C. Development of long-term tolerance versus sensitisation to environmental allergens during the perinatal period. Curr Opin Immunol. 1997;9:782–7. doi: 10.1016/s0952-7915(97)80178-1. [DOI] [PubMed] [Google Scholar]
  • 37.Gammon G, Sercarz EE, Benichou G. The dominant self and the cryptic self. shaping the autoreactive T-cell repertoire. Immunol Today. 1991;12:193–5. doi: 10.1016/0167-5699(91)90052-U. [DOI] [PubMed] [Google Scholar]
  • 38.Adkins B, Du RQ. Newborn mice develop balanced Th1/Th2 primary effector responses in vivo but are biased to Th2 secondary responses. J Immunol. 1998;160:4217–24. [PubMed] [Google Scholar]
  • 39.Woods GM, Qu M, Ragg SJ, Muller HK. Chemical carcinogens and antigens induce immune suppression via Langerhans' cell depletion. Immunology. 1996;88:134–9. doi: 10.1046/j.1365-2567.1996.d01-645.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Niizeki H, Streilein JW. Hapten-specific tolerance induced by acute, low-dose ultraviolet B radiation of skin is mediated via interleukin-10. J Invest Dermatol. 1997;109:25–30. doi: 10.1111/1523-1747.ep12276415. [DOI] [PubMed] [Google Scholar]
  • 41.Ruby JC, Halliday GM, Muller HK. Differential effects of benzo (a) pyrene and dimethylbenz (a) anthracene on Langerhans cell distribution and contact sensitization in murine epidermis. J Invest Dermatol. 1989;92:150–5. doi: 10.1111/1523-1747.ep12276661. [DOI] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES