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The Journal of Immunology, 2006, 176: 2590-2599.
Copyright © 2006 by The American Association of Immunologists

Ceramide-Dependent Regulation of Human Epidermal Keratinocyte CD1d Expression during Terminal Differentiation1

Rita Fishelevich*, Alla Malanina*, Irina Luzina{dagger}, Sergei Atamas{dagger}, Miriam J. Smyth{ddagger}, Steven A. Porcelli§ and Anthony A. Gaspari2,*,¶

* Department of Dermatology, {dagger} Division of Rheumatology, Department of Medicine, and {ddagger} Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201; § Department of Microbiology and Immunology and Department of Medicine, Albert Einstein College of Medicine, New York, NY 10461; and Research Service, Veteran’s Administration Medical Center, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human keratinocytes (KC), when cultured under conditions to remain undifferentiated or to terminally differentiate, changed their cellular distribution of CD1d. As studied by confocal microscopy, undifferentiated KC had a pool of cytoplasmic CD1d, whereas after terminal differentiation, this molecule localized in the cell membrane, which recapitulates CD1d expression in vivo. A comparison of undifferentiated and differentiated cultured KC did not reveal any differences in the association with beta2-microglobulin, invariant chain of class II MHC, or patterns of glycosylation, suggesting that these biochemical properties are not regulating the cellular distribution of CD1d. Time-course studies of CD1d gene expression indicated that KC slowly increased gene expression with CaCl2-induced terminal differentiation. Increased CD1d gene expression was dependent on ceramide synthesis, because fumonisin B1, a ceramide synthetase inhibitor, blocked the increase in CD1d gene expression during terminal differentiation. Similarly, exogenous ceramide or the ceramidase inhibitor, B13, induced CD1d gene expression by undifferentiated, but not terminally differentiated, KC. A protein kinase C-{zeta} (PKC-{zeta}) inhibitor (a pseudosubstrate oligopeptide), but not a PKC-{alpha}beta inhibitor, significantly decreased CD1d gene expression by undifferentiated or ceramide-stimulated cultured, undifferentiated KC. As expected, downstream signaling events of PKC-{zeta} (JNK phosphorylation and NF-{kappa}B accumulation in the nucleus) were also attenuated. The calcineurin phosphatase inhibitor cyclosporine A, which blocks KC terminal differentiation, also blocked CD1d gene expression by cultured KC. In conclusion, this novel function of cellular ceramides extends the importance of this class of biologically active lipids beyond that of terminal differentiation and barrier function in normal human skin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human epidermal keratinocytes (KC)3 are an important component of the skin immune system, because they can influence immune responses in the skin. KC can impact skin-homing lymphocytes by their production of a variety of cytokines (1). The constitutive expression of MHC Ags, class I MHC, as well as the induced expression of class II MHC and adhesion/costimulatory molecules during inflammatory responses allow epidermal KC to directly present Ags to mature, peptide-specific, CD8+ or CD4+ T cells (2, 3).

In addition to the constitutive expression of class I MHC and the induced expression of class II MHC molecules, KC have been noted to express a nonpolymorphic, class I MHC-like molecule, CD1d (4, 5). In contrast to MHC class I or II molecules, which present peptide ligands to the mature peripheral CD8+ or CD4+ T lymphocytes, CD1d presents glycolipids to NK-T lymphocytes (NKT cells). This unique subset of T lymphocytes expresses a T cell {alpha}beta receptor with a restricted repertoire, commonly V{alpha}24 and Vbeta11 in humans (6). They may be CD4/CD8 double negative or CD4+ or CD8+ single positive, and they recognize glycolipids presented by CD1d. NKT cells recognize glycolipids, such as {alpha}-galactosylceramide, and other glycolipids, such as glycosylphosphatidylinositols, derived from self or microbial origins. After activation by glycolipids presented on CD1d, NKT cells have been noted to produce polarizing cytokines, such as IL-4 and IFN-{gamma}, suggesting a role for this cell type in immunoregulation of adaptive immune responses (7).

Although the biologic significance of CD1d expression by epithelial cells has not been well studied, there is evidence that its regulated expression may be relevant to human skin diseases, such as psoriasis (5). In this dermatologic disorder, CD1d expression is increased on the surface KC at all layers of the epidermis and is accompanied by a cellular infiltrate of CD161+ cells in close proximity within the epidermis. This suggests that KC-NKT cell interactions (i.e., the presentation of exogenous (microbial) or endogenous glycolipids) may be relevant to this immune-mediated inflammatory skin disorder.

In addition to epidermal KC in the skin, CD1d is expressed centrally in the thymus and by several other peripheral epithelia or stromal cells in diverse organs such as the intestine, liver, kidney, pancreas, uterus, and conjunctiva (7). Some bone marrow-derived cells, such as B cells and monocyte-derived dendritic cells, also express CD1d (7, 8). Thus, the wide distribution of CD1d in widely divergent tissues by predominantly nonprofessional APCs suggests that it plays an important, yet poorly defined, role in both health and disease states.

In the intestine, the expression of CD1d by epithelial cells is polarized to apical and lateral epithelia in both small and large intestines (4, 9, 10), with a pool of cytoplasmic CD1d in subapical areas of this simple columnar epithelium. In parallel with the polarized expression of CD1d in the gut, there is also an anatomically polarized expression of CD1d in the epidermis of the skin. Although in the gut there is a simple columnar epithelium that interfaces with the environment, in the skin there is a stratified squamous epithelium, in which the outermost layers interface with the environment. Previous studies of normal human skin indicated that CD1d membrane expression by epidermal KC is limited predominantly to the outermost layers of epidermis, immediately below the stratum corneum (5). Although the reasons for this polarized expression in the epidermis have not been identified, it has been hypothesized that the lipid-rich stratum corneum is the microenvironment that may play a role in the compartmentalized expression of CD1d in the membrane of the upper epidermis.

Because epidermal KC are known to increase their synthesis of ceramides during their transit from the basal layer through the stratum corneum (11, 12, 13), we hypothesize that cellular ceramides may modulate CD1d expression in epidermal KC. In this study, we report that human epidermal KC recapitulate the polarized expression of CD1d when cultured under conditions that induce terminal differentiation (i.e., high concentrations of extracellular calcium chloride). Furthermore, this differentiation-induced expression of CD1d by KC can be increased by the addition of extracellular ceramide or the chemical agent B13, a ceramidase inhibitor (14), which induces the accumulation of intracellular ceramide. KC as well as fibroblast CD1d gene expression is inhibited by agents that block sphingolipid synthesis (fumonisin B1) (15). Cyclosporin A, which blocks KC terminal differentiation (16), also inhibited CD1d gene expression. These data suggest that both extracellular and endogenous ceramides modulate CD1d expression and provide a mechanism for the polarized expression of KC CD1d during the differentiation process in vivo within the epidermis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human KC culture

Foreskins were obtained from a newborn nursery with approval of the local institutional review board. The tissue was then stored at 2–8°C until use. Single-cell suspensions of KC were prepared using standard methods (5). KC were cultured in the presence of low (0.05 mM) or high (1.5 mM) concentrations of CaCl2 in the growth medium. The concentration of keratinocytes was determined using a hemocytometer. The primary cells were seeded into T-75 flasks at a cell density of ~3 x 106 cells/flask in 10–15 ml complete medium. The flasks were gassed with 5% CO2 in air and incubated, loosely capped, at 37°C. The flasks were fed fresh complete medium every 2–3 days or passaged into a new T flask.

The human KC cell line HaCat was used in selected experiments. This KC cell line was cultured using standard conditions of DMEM, 10% FCS, and antibiotics (17).

D-Erythro-C6-ceramide (N-hexanoyl-D-erythro-sphingosine) was purchased from Matreya Lipids and was used as a source of ceramide for in vitro studies of cultured KC. Fumonisin B1 (derived from Fusarium moniliforme) was purchased from EMD Biosciences and was used to inhibit ceramide synthesis by cultured human KC during terminal differentiation. The ceramidase inhibitor, B13 (14), was a gift from Dr. A. Bielawska (Medical University of South Carolina, Charleston, SC).

Protein kinase C (PKC) peptide inhibitors

To inhibit PKC-{zeta} activity, a cell-permeable myristoylated pseudosubstrate that included aa 113–125 of the pseudosubstrate region (Myr-SIYRRGARRWRKL-OH; Calbiochem) (18) was added to KC cultures. To inhibit PKC-{alpha}beta enzymatic activity, a myristoylated PKC peptide inhibitor specific for the pseudosubstrate region of PKC-{alpha} and -beta (sequence: Myr-RFARKGALRQKNV; Promega) (19) was added to KC cultures. The peptide inhibitors were used at a concentration of 1–10 µM and were added for a 30-min pulse incubation, then removed by exchanging culture medium.

Protein extraction

Adherent KC were rinsed twice with PBS buffer after removing the growth medium, and protease inhibitor mixture was added to the mixture (1/100 dilution). Cell lysis buffer (radioimmunoprecipitation assay (RIPA)) was added (1 ml/106-107 cells) for 15 min on an orbital shaker. The cell lysate was collected by scraping, and the lysed cells were centrifuged for 10 min at 12,000 x g. All of the above steps were performed at 2–8°C. The proteins concentration was measured using Bio-Rad protein assay. 20–30 µg of proteins were loaded on the 10% Bis-Tris NuPage (denaturing) gel for electrophoresis. Gels were transferred to nitrocellulose membrane with 10% methanol and analyzed by WesternBreeze Chemiluminescent detection kit (Invitrogen Life Technologies).

Immunoprecipitation and immunoblotting

After removing all culture medium, the cells were washed twice with ice-cold PBS. The cells were incubated on ice for 10–15 min after adding lysis buffer to a concentration of 106–107 cells/ml, then centrifuged at 12,000 x g for 10 min at 4°C. One milliliter of Protein G-Sepharose 4 Fast Flow beads (Immunoprecipitation Starter Pack; Amersham Biosciences) were washed three times in RIPA by centrifugation at 12,000 x g for 20 s. Twenty microliters of the washed beads were transferred to the tube containing the cell lysate. The mixture was incubated on the rocker for 30 min at 4°C. The beads were then removed by centrifugation at 12,000 x g for 20 s. To the lysate, 1–5 µg of CD1d51 was added. The mixture was incubated overnight on the rocker at 4°C. The Ag-Ab-bead complexes were washed four times in 500-1000 µl of ice-cold RIPA and centrifuged at 12,000 x g for 20 s. After the final wash, the samples were resuspended in 30 µl of denaturing buffer, followed by 95°C for 5 min, and centrifugation at 12,000 x g for 20 s (to remove beads). The supernatant was run on 10% bis-Tris NuPage gel for electrophoretic analysis.

To detect phosphorylation of JNK in KC, 1 x 106 cells were lysed with lysis buffer (RIPA buffer with a Phosphatase Inhibitor Cocktail II; Sigma-Aldrich) scraped from the culture vessel. Cellular debris was pelleted and removed, and protein concentration was determined (Bradford method). The lysate was then run on a 10% bis-Tris NuPage gel under reducing conditions with MOPS-SDS running buffer. The separated proteins were transferred to a nitrocellulose membrane and stained with a rabbit polyclonal Ab specific for JNK. Staining was detected using Western Breeze Chemiluminescent Detection kit (Invitrogen Life Technologies); a Chemdoc (Bio-Rad) digital imaging system was used to record the resulting images.

To detect nuclear NF-{kappa}B, lysis buffer (Panomics) was added to 107 adherent KC for 10 min on ice, followed by scraping and pipetting to disaggregate clumps. The extract was centrifuged at 15,000 x g for 3 min, and the supernatant was discarded. The pellet was resuspended in buffer B mix, followed by vortexing. This was then centrifuged again at 15,000 x g for 20 min, and the supernatant was collected (nuclear extract). The lysate was used for Western blotting, using the methods described above. The mAb F-6 (monoclonal mouse IgG1 specific for p65 subunit of human NF-{kappa}B; Santa Cruz Biotechnology) was used to probe for NF-{kappa}B in nuclear extracts.

Endonuclease digestion of the immunoprecipitated sample (peptide-N-glycosidase F (PNGase F)

Twenty micrograms of glycoprotein was denatured in 1x glycoprotein denaturing buffer at 100°C for 10 min. Buffer was added followed by 1–5 µl of PNGase F (New England Biolabs). The mixture was incubated at 37°C overnight. After the incubation, loading buffer was added, and the mixture was boiled for 5 min, followed by centrifugation at 12,000 x g for 20 s. The supernatant was run on 10% bis-Tris NuPage gel for electrophoretic analysis.

RNA extraction and cDNA synthesis (RNeasy Mini Protocol; Qiagen)

Cultured KC (from 1 well of a 6-well plate) were lysed by adding 350 µl of beta-ME in standard RNeasy buffer (1/100) directly into the culture vessels. The sample was then homogenized by placing it into a QIAshredder spin column and centrifuging for 2 min at maximum speed. Seventy percent ethanol (350 µl) in diethyl pyrocarbonate H2O was added to the homogenized lysate and mixed well. The 700-µl sample was placed in an RNeasy mini column and centrifuged for 15 s at ≥10,000 rpm, and the flow-through was discarded. The RNA was eluted with 30 µl of RNase-free water. Synthesis of cDNA was completed using standard methods (20).

Real-time PCR

Real-time PCR (LightCycler; Roche) was used to confirm differences in levels of expression of selected mRNA. The primers, PCR protocol, and product quantification for 18S ribosomal RNA were exactly as reported previously (21). All other primers and hybridization probes were designed and prepared by ITB Molbiol. The hybridization probes were labeled with fluorescein at the 5' terminus (3FL) and with LightCycler Red at the 5' terminus of the other probe. Amplification of a single PCR product was confirmed by gel electrophoresis and melting curve analyses. The sequences of the sense, antisense, and internal hybridization probes are as follows: 18S sense, 5'-AACCCGTTGAACCCCATT-3'; 18S antisense, 5'-CCATCCAATCGGTAGTAGCG-3'; CD1d (sense), 5'-AGACATGGTATCTCCGAGCAAC-3'; antisense, 5'-CTGAGCAGACCAGGACTGAA-3'; FL-probe, 5'-TA+GTAGCTCCCACCCCAGTAGAGGAC-FL-3'; and LC-probe, 5'-LC Red640-ATGTCCTGGCCCTCTAGACTGCTGTG-PH-3'. After denaturation, the cDNA were subjected to 40–50 cycles of amplification, followed by melting curve analysis.

Polymerase chain reaction

Detection of CD1d mRNA was performed using RT-PCR. Sequences of the primers were as follows: exon 2 sense, 5'-CTG CAG ATC TCG TCC TTC GCC AAT-3'; and exon 3 antisense, 5'-TTG AAT GGC CAA GTT TAC CCA AAG-3'. These primers amplified a 400-bp product using an annealing temperature of 55°C and 35 cycles of PCR. The PCR products were run on an agarose gel and photographed. These primers have been demonstrated to be specific for CD1d and do not amplify CD1a, CD1b, or CD1c (5).

Staining of adherent or trypsinized cell suspensions

KC were cultured on sterile coverslips in medium as described above. The adherent cells were permeabilized for staining by incubation with 100% methanol for 10 min at 4°C, then they were rinsed extensively with PBS-0.1% BSA-1% FCS. Primary Abs NOR3.2 (1/100 dilution in PBS-0.1% BSA-1% FCS) were added to the fixed cells and allowed to react for 1 h at room temperature. The cells were then incubated at 4°C for 1 h with FITC-conjugated goat anti-mouse IgG (1/100 dilution in PBS-0.1% BSA). PermaFluor Aqueous Mounting Medium (Immunon) was used to mount the cells to the slide. The cells were washed with PBS after each step. For routine fluorescence microscopy, the stained cells were examined with a Nikon Eclipse E600 epifluorescence microscope equipped with a digital camera (RT-spot slider; Diagnostic Instruments). For laser scanning confocal microscopy, the stained cells were examined with an LSM 510, Axiovert 100 laser confocal microscope (using an argon 458-nm laser for green fluorescence; Zeiss).

For flow cytometry, 1 x 106 KC were trypsinized and centrifuged at 3000 rpm for 5 min. After washing with PBS, 100 µl of ice-cold methanol was added, and the mixture was allowed to react for 10 min on ice. The mixture was divided into two samples. To the experimental sample, primary Abs (monoclonal anti-CD1d (NOR 3.2)) at a concentration of 1/50 in PBS-0.5% BSA-1% FCS were added; to the control sample, only PBS-0.5% BSA-1% FCS was added. The samples were incubated on ice for 1 h and washed with PBS. Secondary Abs (goat anti-mouse IgG) at 1/100 PBS-0.5% BSA-FITC were added and incubated for 1 h on ice. After washing, the cells were resuspended in 500 µl of PBS-0.5% BSA-1% FCS. The cell suspensions were then analyzed by a Coulter EPICS Elite ESP flow cytometer. Viable KC were identified by forward and side scatter properties, with dead cells excluded from analysis. A total of 50,000 events from each sample was analyzed.

Monoclonal Abs

The following mAbs were used in these studies: anti-CD1d: clone CD1d51 (immunoprecipitation); clone CD1d75 (Western blotting) (22) and Nor 3.2 (staining of adherent cells; in situ immunochemistry, and flow cytometry; BioSource International); anti-CD74 (invariant chain of class II MHC): clone LN-2 (Santa Cruz Biotechnology), rabbit antiserum to human beta2-microglobulin (beta2m) (Western blotting; Research Diagnostics), mouse mAb to human B2 HLA-A, -B, and -C (clone B-2; gift from Dr. H. Plough, Harvard Medical School, Boston, MA); and mouse mAb to human involucrin (clone SY5; Research Diagnostics).

Statistical analyses

Quantitative data were analyzed for statistically significant differences between control and treatment groups using the GraphPad Instat software. Because multiple comparisons were examined, a one-way ANOVA was applied to the quantitative data (p < 0.05 was considered significant).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Previous studies of CD1d expression in normal and uninvolved psoriatic skin suggested localization of this molecule to the outermost layers of the skin in the lipid-rich stratum granulosum (4, 5). The highest expression of epidermal KC CD1d was in the outer epidermis, which colocalized with involucrin staining, a biochemical marker of KC terminal differentiation (21). This colocalization was observed in foreskin (data not shown) and adult human skin (Fig. 1A). To determine whether cultured human KC can recapitulate this apparent linkage of CD1d expression with terminal differentiation, primary human KC monolayers (undifferentiated or differentiated; see Materials and Methods) were fixed with methanol, stained with Nor3.2 mAb and secondary Ab, and studied for their cellular distribution of CD1d using confocal laser scanning microscopy. Although terminally differentiated KC demonstrated bright staining of the cellular membrane with CD1d (Fig. 1B, left panel), in undifferentiated KC, CD1d was localized to the cytoplasm, with an absence of any apparent membrane staining (Fig. 1B, right panel). Cell suspensions of undifferentiated or terminally differentiated KC also recapitulated these findings (data not shown). To confirm these immunofluorescence findings of normal human skin and cultured monolayers, lysates were prepared from undifferentiated KC, terminally differentiated KC (24 h in serum-free medium containing 1.2 mM CaCl2), and Jurkat cells (an immortalized T cell line that is CD1d positive) and Western blotted for CD1d (CD1d75 mAb) and, in parallel, involucrin (see Materials and Methods). Both undifferentiated and terminally differentiated KC (24 h in 1.2 mM CaCl2), and Jurkat cells all expressed the 50-kDa isoform of CD1d (Fig. 1C). In contrast, only terminally differentiated KC expressed significant involucrin, consistent with previous the literature about cellular differentiation and involucrin expression (23). Normalization of total protein loaded vs densitometry scanning of CD1d-stained bands on nylon membranes of the Western blot indicated that 24-h terminally differentiated and undifferentiated KC expressed a similar CD1d Ag content, which was much less than that of Jurkat cells (data not shown).


Figure 1
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  		FIGURE 1. Characterization of KC CD1d in vivo and in vitro. A, Cryostat sections of normal human adult skin were double stained with Abs specific for CD1d (green) and involucrin (red) or the appropriate isotype-matched negative controls, and photomicrographs were taken. Yellow staining represents KC coexpressing CD1d and involucrin (x40 magnification). B, Confocal microscopy of cultured, plastic-adherent, differentiated or undifferentiated KC permeabilized with ethanol and stained with Nor 3.2 (anti-CD1d), followed by a fluoresceinated secondary reagent (x40 magnification). C, Lysates were prepared from cultured, plastic-adherent, differentiated or undifferentiated KC or the nonadherent T cell line, Jurkat cells. Using standard techniques, these lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes for staining with anti-CD1d (CD1d75 mAb) or involucrin. D, Time course of CD1d expression during CaCl2-induced terminal differentiation. Lysates were prepared from undifferentiated KC or differentiated KC at the indicated times after adding high concentrations (1.20 mM) of extracellular CaCl2. Top panel, Western blot; bottom panel, digital image analysis of pixel density of Western blot (normalized to the total protein added to each lane).

 
Cultured KC up-regulate their expression of CD1d late during terminal differentiation

To study the effects of CaCl2-induced terminal differentiation on the kinetics of CD1d expression, lysates were prepared from undifferentiated KC (culture medium containing 0.05 mM CaCl2) and from the same set of KC that were terminally differentiated (24, 48, and 72 h after culture in medium containing 1.20 mM CaCl2) and Western blotted with the anti-CD1d mAb, CD1d75 (Fig. 1D). After 24 h of terminal differentiation, there was no appreciable change in CD1d Ag content compared with undifferentiated KC; however, after 48 and 72 h, there was an increase in CD1d Ag content (Fig. 1D; depicted data represent adjusted pixel volume, normalized to total protein per sample). This increase in CD1d expression associated with terminal differentiation of human KC was reproduced with three different sets of cultured KC (data not shown).

Membrane expression of KC-derived CD1d is not differentially regulated by association with beta2m, class II MHC invariant chain (Ii), or glycosylation patterns

To better understand why differentiated KC express membrane-associated CD1d and undifferentiated KC express cytoplasmic CD1d, biochemical characterization of CD1d was performed. There is evidence that the association of class Ia MHC molecules with beta2m is necessary for membrane expression, and those MHC that are expressed on the cell surface independently of beta2m are nonfunctional (24, 25, 26, 27, 28). Additionally, beta2m gene-targeted mice express very low levels of class I MHC (29). However, in the human intestinal epithelium, class Ib MHC (CD1d) can be expressed in the absence of an association with beta2m (30). We hypothesized that there may be differences in the association of CD1d with beta2m in undifferentiated and differentiated KC, with a possible lack of association in undifferentiated KC and an association with differentiated KC. Thus, the association of CD1d with beta2m in differentiated or undifferentiated KC was examined (immunoprecipitation of class I MHC or CD1d, followed by immunoblotting for beta2m). CD1d was associated with beta2m in both differentiated and undifferentiated KC (Fig. 2A). These data suggest that differential association of CD1d with beta2m during terminal differentiation is not responsible for the localization of this molecule in the cytoplasm in undifferentiated KC or its localization in the membrane of terminally differentiated KC.


Figure 2
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  		FIGURE 2. CD1d expressed by undifferentiated KC does not differ biochemically from that of differentiated KC. A, KC association of beta2m was examined by immunoprecipitating (IP) lysates from undifferentiated or differentiated KC with anti-HLA-A, -B, and -C (clone B2) CD1d (clone CD1d51) or an irrelevant IgG, followed by immunoblotting with a rabbit antisera specific for human beta2m. B, KC CD1d glycosylation patterns were examined by studying lysates from adherent HaCat, undifferentiated KC, and differentiated KC and immunoprecipitated with anti-CD1d (clone CD1d51). Then they were left untreated or were digested with PNase F, subjected to SDS-PAGE, and Western blotted with anti-CD1d (clone CD1d75). C, Association of KC CD1d with the class II Ii was studied by immunoprecipitating lysates from adherent-cultured, undifferentiated or differentiated KC with anti-CD1d (clone CD1d51), then immunoblotting the precipitates with anti-CD74.

 
To determine whether differences in glycosylation patterns of CD1d by differentiated or undifferentiated KC influenced cytoplasmic or membrane trafficking of this molecule (31), PNGase F digestion of CD1d was studied (Fig. 2B). PNGase F digestion of KC-derived CD1d in the HaCat KC cell line and differentiated and undifferentiated KC resulted in a change in the apparent molecular mass to 30 kDa (from ~50 kDa in undigested immunoprecipitates of these same cell types). These data suggest that differences in glycosylation of CD1d in differentiated and undifferentiated KC are not responsible for membrane vs cytoplasmic localization.

Because CD1d is known to associate with the class II MHC Ii in the cytoplasm of APCs (32), its association with Ii in control (class II MHC-negative) and IFN-{gamma} treated (class II MHC-positive) undifferentiated and differentiated KC was studied. CD1d was immunoprecipitated from class II MHC-negative or -positive primary human KC or class II MHC-positive Jurkat cells, then the complex was dissociated, separated by SDS-PAGE, and immunoblotted with an mAb specific for CD74 (Ii). Surprisingly, CD74 was associated with CD1d in control, class II MHC-negative, differentiated and undifferentiated KC as well as class II MHC bearing (IFN-{gamma} treated) differentiated and undifferentiated KC (Fig. 2C). There was no differential association of Ii with CD1d in class II-negative or -positive (IFN-{gamma}-treated) KC. These data suggest that Ii association with CD1d in KC occurs independently of the state of differentiation as well as their expression of cell surface class II MHC and that association of CD1d with Ii does not play a role in cell surface expression during KC during terminal differentiation.

CD1d gene expression increases during terminal differentiation

KC CD1d gene expression was studied in undifferentiated and differentiated KC using RT-PCR. As depicted in Fig. 3A, undifferentiated and differentiated KC from two different donors expressed CD1d transcripts. Using real-time PCR, the kinetics of CaCl2-induced terminal differentiation of KC on CD1d gene expression were studied over 48 h. After 12 h of culture in high 1.20 mM CaCl2, there was a significant increase in steady-state mRNA encoding CD1d, which peaked at 24 h (Fig. 3B). In other experiments, it was determined that by 72 h, CD1d gene expression began to decrease from its peak at 24 h (data not shown).


Figure 3
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  		FIGURE 3. CaCl2-induced terminal differentiation of cultured human KC induces CD1d gene expression. A, RT-PCR to amplify CD1d cDNA from two sets of undifferentiated and differentiated KC. B, Real-time PCR to study the time course of CD1d gene expression after CaCl2-induced terminal differentiation of cultured human KC. *, p < 0.01 compared with control (by one-way ANOVA). Data represent the mean ± SD of triplicate samples. Results are from a single representative experiment that was repeated three times. M, markers.

 
Exogenous ceramide as well as cellular ceramide synthesis modulate CD1d gene expression in cultured human KC

Because CD1d expression increases during terminal differentiation, and KC are known to increase their production of ceramides during the differentiation process (13), we hypothesized that ceramides themselves may influence KC CD1d gene expression. When undifferentiated KC were cultured in the presence of 0.5–10 µM exogenous ceramides, CD1d gene expression increased ~2-fold during a 6-h incubation with this molecule (Fig. 4A). In contrast, terminally differentiated KC were resistant to the effects of the addition of exogenous C6-ceramide on CD1d gene expression (data not shown). Additionally, B13, a chemical agent that inhibits cellular ceramidase activity (14), resulting in the intracellular accumulation of ceramide, also increased KC CD1d gene expression in undifferentiated, but not differentiated (Fig. 4B) KC. Assays of total cellular ceramide content of B13-treated KC exhibited elevated ceramide levels compared with control KC (data not shown).


Figure 4
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  		FIGURE 4. Peturbations in cellular ceramide levels regulate CD1d gene expression in cultured human KC. A, Undifferentiated KC were cultured in medium alone, medium containing a vehicle, and medium containing increasing concentrations of extracellular C6-ceramide for 6 h. After this, mRNA was extracted, cDNA was synthesized, and CD1d gene expression was studied using real-time PCR. *, p < 0.05 compared with control (by one-way ANOVA). B, Effects of the ceramidase inhibitor, B13, on undifferentiated and differentiated KC CD1d gene expression. *, p < 0.01 compared with control (by one-way ANOVA). Data represent the mean ± SD of triplicate samples. Results are from a single representative experiment that was repeated three times.

 
Because ceramide synthesis is known to be active in cultured human KC, we asked whether ceramide synthesis regulated CD1d gene expression in cultured human KC. The effect of inhibition of the de novo pathway of ceramide synthesis was examined using the sphinganine-N-acyltransferase (ceramide synthetase) inhibitor, fumonisin B1 (15). KC cultured in the presence of fumonisin B1 exhibited a significantly impaired response to differentiation-induced increase in CD1d gene expression (Fig. 5A). These gene expression data were confirmed by flow cytometry of trypsinized cell suspensions of ethanol-permeabilized undifferentiated or differentiated normal human KC, in which fumonisin B1 decreased the frequency of CD1d-expressing KC in both undifferentiated and differentiated KC (Fig. 5B). These data suggest that fumonisin B1 inhibition of de novo ceramide synthesis inhibits CD1d gene expression as well as cytoplasmic or membrane-associated CD1d in undifferentiated as well as differentiated KC, respectively. To determine whether the fumonisin effect was specific for CD1d gene expression or whether it inhibited other events associated with terminal differentiation, involucrin expression was studied by Western blotting in fumonisin-treated KC (Fig. 5C). Fumonisin-treated KC exhibited a slight decrease in involucrin expression (not significantly different from control KC), indicating that CD1d gene expression is more sensitive to inhibition of ceramide synthesis than are other cellular differentiation events. Assays of total cellular ceramide content confirmed that fumonisin-treated KC exhibited diminished ceramide content compared with control KC (data not shown).


Figure 5
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  		FIGURE 5. The ceramide synthetase inhibitor, fumonisin, inhibits CD1d gene expression in KC and dermal fibroblasts. A, The effect of fumonisin on CD1d gene expression during CaCl2-induced terminal differentiation. KC were cultured in medium containing low concentrations of CaCl2 (0.05 mM) in the absence or the presence of the indicated concentrations of fumonisin for 24 h before adding high concentrations of CaCl. Twenty-four hours after switching the medium, mRNA was extracted, cDNA was synthesized, and real-time PCR was run to assay CD1d gene expression. *, p < 0.01 compared with control (by one-way ANOVA). B, Trypsinized cell suspensions of ethanol-permeabilized KC (untreated or fumonisin B1 treated) were studied for CD1d expression using flow cytometry and staining with the mAb Nor 3.2. C, Western blotting to study the effects of fumonisin on the keratinocyte differentiation marker, involucrin. D, Exogenous ceramide prevents fumonisin B1 inhibition of KC CD1d during terminal differentiation. *, p < 0.05 compared with control (by one-way ANOVA). E, Fumonisin inhibits CD1d gene expression in dermal fibroblasts. *, p < 0.05 compared with control (by one-way ANOVA). Data represent the mean ± SD of triplicate samples. Results are from a single representative experiment that was repeated three times.

 
To determine whether exogenous ceramide would reverse the fumonisin B1 inhibition of KC CD1d expression during terminal differentiation, undifferentiated KC were incubated with fumonisin B1 and ceramide in combination (Fig. 5D). Exogenous ceramide rendered KC resistant to fumonisin B1 inhibition of CD1d gene expression during terminal differentiation, indicating the critical role of ceramide in regulating CD1d gene expression by cultured human KC. To determine whether this phenomenon is specific for KC, dermal fibroblasts were incubated in medium alone or medium containing IFN-{gamma} in the presence of increasing doses of fumonisin, and CD1d gene expression was studied (Fig. 5E). This agent inhibited CD1d gene expression in resting or IFN-{gamma}-treated fibroblasts, indicating that ceramide synthesis modulates CD1d expression in cells other than KC.

To test our hypothesis that basal and ceramide-stimulated CD1d gene expression in cultured human KC is regulated by PKC signal transduction pathways, we cultured KC in the presence of myristoylated oligopeptide pseudosubstrates that inhibit either PKC-{alpha}beta or PKC-{zeta} (18, 19) (Fig. 6, A and B). Only those KC (either unstimulated or ceramide stimulated) cultured with peptide pseudosubstrate PKC-{zeta} inhibitor exhibited a profound decrease in CD1d gene expression (p < 0.01 compared with control for both doses of inhibitor, highly significant). The peptide pseudosubstrate PKC-{alpha}beta inhibitor had no effect on basal or ceramide-stimulated CD1d gene expression. These data suggest that PKC-{zeta}, an atypical PKC isoform that is ceramide dependent (33, 34, 35), plays a role in regulating CD1d gene expression in human KC. Because PKC-{zeta} signaling has been demonstrated to activate MAPK (36), phosphorylation of JNK was studied by Western blotting in control KC or those treated with PKC-{zeta} or PKC-{alpha}beta pseudosubstrate inhibitors (Fig. 6C). Only those KC treated with the PKC-{zeta} inhibitor exhibited a significant decrease in steady-state phosphorylated JNK. Similarly, nuclear NF{kappa}B accumulation, a terminal event associated with PKC-{zeta} signaling (Fig. 6D), was decreased only in those KC treated with the PKC-{zeta} inhibitor. The inhibition of CD1d gene expression by PKC-{zeta} was confirmed by Western blotting (Fig. 6E), confirming that blocking the enzymatic activity of this ceramide-dependent enzyme inhibited CD1d at the protein level.


Figure 6
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  		FIGURE 6. Basal and ceramide-stimulated CD1d gene expression in cultured KC is dependent on PKC-{zeta}, but not PKC-{alpha}beta, enzymatic activity. A synthetic oligopeptide that serves as a pseudosubstrate inhibitor for PKC-{zeta} or PKC-{alpha}beta was added to cultured human KC in the absence (A) or the presence of 10 µM ceramide (B) for 30 min, then removed by washing. RNA was extracted 6 h later to study CD1d gene expression by real-time PCR. Data represent the mean ± SD of triplicate samples. Results are from a single representative experiment that was repeated three times. KC were pulse-incubated with medium alone or medium containing PKC-{zeta} or PKC-{alpha}beta inhibitors; 30 min later, the cells were lysed, and JNK phosphorylation (C) or nuclear NF-{kappa}B content (D) was studied by Western blotting. E, KC were pulse-incubated with the indicated concentrations of the pseudosubstrate inhibitors of PKC-{zeta} or PKC-{alpha}beta enzymatic activity for 30 min; 48 h later, a lysate was prepared, and CD1d content was studied with Western blotting.

 
The calcineurin inhibitor, cyclosporin A, inhibits the increase in CD1d associated with terminal differentiation

KC have been demonstrated to increase their translocation of NFAT into the nucleus, in vivo during normal differentiation, in psoriatic skin lesions, as well as in vitro after exposure to agonists that induce terminal differentiation (increased extracellular CaCl2 or 12-O-tetradecanoyl-phorbol-13-acetate plus ionomycin) (16). Cyclosporin A is an immunosuppressive agent that blocks calcineurin phosphatase in T lymphocytes as well as KC (16). As such, this agent has been demonstrated to block the KC response to differentiating agents, as described above. Thus, cyclosporin A treatment of KC, which blocks their terminal differentiation induced by high extracellular concentrations of CaCl2 (1.2 mM), would be expected to inhibit CD1d if the regulation of this molecule were indeed linked with KC terminal differentiation. KC cultured in medium with 5 or 10 µg/ml cyclosporin A exhibited a blunted increase in CD1d gene expression compared with control KC after exposure to the differentiating signal of increased extracellular CaCl2 (1.2 mM; Fig. 7A). These gene expression data were confirmed by flow cytometry analysis of trypsinized single-cell suspensions of ethanol-permeabilized, differentiated KC (Fig. 7B). These data suggest that NFAT, a transcription factor associated with KC differentiation, directly or indirectly plays an important role in regulating CD1d gene expression. Similar to fumonisin-treated KC, cyclosporin A-treated KC exhibited a diminished ceramide content compared with control cells (data not shown).


Figure 7
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  		FIGURE 7. Effect of cyclosporin A on CD1d gene expression in differentiated KC. A, Undifferentiated KC were treated with 5 or 10 µg/ml cyclosporin A for 24 h, then switched to medium containing 0.05 or 1.2 mM CaCl2 for 24 h. Then mRNA was extracted, cDNA was synthesized, and real-time PCR was completed to study the relative gene expression of CD1d. **, p < 0.001 compared with control (by one-way ANOVA). Data represent the mean ± SD of triplicate samples. Results are from a single representative experiment that was repeated three times. B, Flow cytometry to study CD1d expression in differentiated KC cultured in medium alone (control) or medium containing cyclosporin A.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In normal human skin, as KC move from the basal layer of the epidermis, they undergo a process of terminal differentiation in which they change their morphology, lose their ability to proliferate, cross-link polypeptides associated with their plasma membrane, secrete lipids into the extracellular space, and gradually lose their nucleus. All these events are necessary for formation of the stratum corneum, which is responsible for the barrier function of the skin (37). When KC are cultured in monolayers in serum-free medium, they recapitulate some components of this differentiation program in response to changes in extracellular concentrations of CaCl2, which is thought to mimic the CaCl2 gradient in the epidermis during terminal differentiation (38).

Consistent with previous reports of the polarized expression of CD1d in the upper epidermis of normal human skin (4, 5), our double staining of normal human skin with a marker of terminal differentiation, involucrin, indicates that expression is maximal in the upper epidermis (37, 38). Similarly, when human KC are cultured in vitro under conditions to maintain them in an undifferentiated state (0.05 mM extracellular CaCl2) or induce them to terminally differentiate (1.20 mM extracellular CaCl2), CD1d is modulated (cytoplasmic localization in undifferentiated KC; membrane expression in terminally differentiated KC; Fig. 2). This also recapitulates the events that occur in vivo in the epidermis (Fig. 1). Thus, cultured human KC represent an excellent model for study of the regulation of CD1d gene expression and its association with the cell membrane. Such studies of the regulation of CD1d gene expression by KC may provide insights into its overexpression in dermatologic diseases such as psoriasis. In this disease, KC at all layers of the epidermis express CD1d, and there is a cellular, intraepidermal infiltrate of NKT cells (5), which may be relevant to the pathogenesis of this common, immune-mediated, inflammatory skin disorder. There are a number of other lymphocyte-mediated skin disorders in which KC are induced to express class II MHC (including psoriasis) (39). Thus, it is possible that overexpression of CD1d by KC and NKT cells may play a broad role in contributing to the pathophysiology of a number of different inflammatory skin diseases.

Class I MHC Ag expression on the cell surface is controlled by its association with beta2m (24, 25, 26, 27, 28, 29). It has been demonstrated that in human intestine, CD1d may be expressed at the cell surface in the absence of an association with beta2m (30). In contrast, studies of CD1a, CD1d, and murine CD1.1 indicated that membrane expression of these molecules is dependent on association with beta2m (40, 41, 42, 43, 44). Thus, there appears to be site-specific regulation of CD1d expression by its association with beta2m, with the gut being a site of beta2m-independent expression of CD1d by epithelial cells. This raised the interesting possibility of beta2m regulating KC CD1d membrane expression during terminal differentiation. However, immunoprecipitation and immunoblotting studies did not demonstrate any regulatory role, because beta2m was equally associated with CD1d in undifferentiated KC (cytoplasmic localization) as well as differentiated KC (membrane association).

In addition to its association with beta2m, CD1d has been demonstrated to interact with the invariant-associated HLA-DR molecules (MHC class II Ii) in the endoplasmic reticulum, and this complex is thought to lead to a pathway into late endosomes/lysosomes and the plasma membrane (32, 45). Although the true physiologic significance of the CD1d-Ii association remains to be determined, the study of its role in transport to the plasma membrane in KC provided an opportunity to clarify its role in CD1d-bearing epithelial cells. Immunoprecipitation/immunoblotting studies did not demonstrate any differences between undifferentiated and differentiated KC, in which there is cytoplasmic and membrane localization of CD1d, respectively. In both cell types, CD1d was associated with Ii, independently of cell surface expression of CD1d or even class II MHC (after IFN-{gamma} treatment of undifferentiated or differentiated KC; Fig. 3C). These data suggest that Ii association with CD1d in KC does not play a role in membrane transport.

Cyclosporin A is a fungal polypeptide that is immunosuppressive because it blocks calcineurin phosphatase activity and prevents the translocation of NF of activated T cells (NFAT), hence interfering with T cell activation and cytokine gene transcription (46). This agent has been demonstrated to be an effective therapy for treatment-resistant psoriasis (46), presumably because of its inhibitory effects on T cell activation. Al-Daraji et al. (16) have demonstrated that cyclosporin A also blocks KC nuclear translocation during terminal differentiation. The observation that cyclosporin A blocks KC CD1d expression during terminal differentiation is consistent with our hypothesis that CD1d is linked with KC terminal differentiation, and that the transcription factor NFAT may be involved directly or indirectly with the regulation of CD1d gene expression. The inhibitory effects of cyclosporin A on KC CD1d also suggest a novel mechanism of action of this agent in psoriasis, i.e., the inhibition of CD1d-mediated Ag presentation by KC (because of diminished expression of this molecule) to NKT cells.

Previous studies have demonstrated a role for cytokines such as IFN-{gamma} in regulating cell surface expression of CD1d by professional APCs (47) as well as by cultured KC and presumably in psoriatic skin lesions (5). Our observation that KC increase CD1d gene expression during terminal differentiation (Fig. 4) is the first demonstration that increased CD1d gene expression occurs in the absence of exogenous cytokines. However, it is possible that KC may alter cytokine expression with terminal differentiation and modulate CD1d by an autocrine mechanism. This increase in CD1d gene expression may, in part, be responsible for the increase in membrane expression during KC differentiation.

Addition of exogenous ceramide to undifferentiated KC causes these cells to rapidly (within 6 h) increase CD1d gene expression (Fig. 5, A and B). Although this exogenous ceramide may simply reflect the ability of this class of lipids to induce KC cell cycle arrest and terminal differentiation (48, 49, 50, 51), there is evidence that ceramides can modulate the expression of MHC class II molecules as well as adhesion molecules induced by cytokines such as IFN-{gamma} (52). Wakita et al. (52) demonstrated that inhibitors of sphingolipid synthesis (such as fumonisin B1 and cycloserine) blocked the ability of IFN-{gamma} to induce the expression of the class II MHC molecule, HLA-DR, and the adhesion molecule, ICAM-1, in cultured human KC. This blockade was reversed by the addition of exogenous ceramide, suggesting that ceramide functioned as a modulator of cytokine signaling in KC.

The demonstration that fumonisin B1 blocked the increase in gene expression and total cellular CD1d Ag expression, which was reversible by the addition of exogenous ceramides (Fig. 5, C–E) in cultured KC in response to increased extracellular CaCl2 concentrations, indicates that ceramide synthesis is necessary for the differentiation-associated increase in CD1d gene expression by KC. Similarly, our studies of cultured human fibroblasts (Fig. 5E) and the monocyte cell line THP-1 (data not shown) indicate that that ceramide modulates CD1d in all cell types tested.

The demonstration that the ceramidase inhibitor B13 (resulting in a net increase in the cellular pool of ceramide) (14) increases KC CD1d in undifferentiated KC is also consistent with the hypothesis that cellular ceramide regulates CD1d gene expression. Inhibition of the enzymatic activity of PKC-{zeta}, which is thought to be a ceramide-dependent PKC isoform (32, 33, 34), inhibits JNK phosphorylation, nuclear NF-{kappa}B accumulation (35), and, ultimately, CD1d gene expression (Fig. 6). This represents additional evidence of the important role of ceramide-dependent signaling pathways in controlling CD1d expression. These data suggest that modulating ceramide levels in KC during in vitro differentiation affects the transcriptional regulation of an immunologically relevant molecule that controls immune recognition by NKT cells.

In conclusion, cultured KC are an excellent model with which to better understand the in situ regulation of CD1d by epidermal KC in normal skin and its overexpression in inflammatory skin diseases such as psoriasis (5). This is the first demonstration that CD1d expression in KC and other cell types is regulated by ceramide synthesis, which is known to increase during terminal differentiation. It also presents a new pharmacologic target for regulating CD1d expression in epithelia and by professional APCs and, hence, the immune responses mediated by CD1d-restricted NKT cells, which are recognized as playing an important role in host defense against cancer, infections as well as modulating autoimmune responses in the skin and other tissues.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R01AR46108-05 (to A.A.G.). Back

2 Address correspondence and reprint requests to Dr. Anthony A. Gaspari, 405 West Redwood Street, 6th Floor, Baltimore, MD 21201. E-mail address: agasp001{at}umaryland.edu Back

3 Abbreviations used in this paper: KC, keratinocyte; beta2m, beta2-microglobulin; Ii, invariant chain; PKC, protein kinase C; PNGase, peptide-N-glycosidase F; RIPA, radioimmunoprecipitation assay. Back

Received for publication February 14, 2005. Accepted for publication November 21, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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