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Production by NK-T Cells1
*
Department of Pathology, Loyola University Medical Center, Maywood, IL 60153;
Department of Dermatology, Institut National de la Santé et de la Recherche Médicale, Unité 346, Hopital Edouard Herriot, Lyon, France;
Department of Dermatology, University of California School of Medicine, Los Angeles, CA 90024; and
§
Department of Medicine, Albert Einstein College of Medicine, New York, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Combining CD1d-positive keratinocytes
with human NK-T cell clones resulted in clustering of NK-T cells, and
while no significant proliferation ensued, NK-T cells became activated
to produce large amounts of IFN-. We conclude that CD1d can be
expressed in a functionally active form by keratinocytes and is
up-regulated in psoriasis and other inflammatory dermatoses. The
ability of IFN- to enhance keratinocyte CD1d expression and the
subsequent ability of CD1d-positive keratinocytes to activate NK-T
cells to produce IFN-, could provide a mechanism that contributes to
the pathogenesis of psoriasis and other skin
disorders. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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can trigger the appearance of psoriatic lesions in genetically
susceptible individuals (2). Most studies of the genetic
factors associated with psoriasis point to chromosome 6 and the class I
MHC as disease-associated loci, including a specific association with
HLA-Cw6 (3, 4) Typically, APCs present peptide Ags in the
context of such MHC class I molecules to CD8+ T
cells (5), but definitive pathological roles for
CD8+ and CD4+ T cells in
psoriasis remain uncertain. Recently, we demonstrated the presence of NK-T cells in the epidermis of acute and chronic psoriatic plaques (6, 7, 8). A hallmark of NK-T cells is their expression of certain C-type lectin NK cell receptors (NKRs)4 such as CD94 and CD161 (9, 10, 11). Classical NK-T cells may plan an immunoregulatory role for recognition of both self and foreign Ags and are implicated in the pathogenesis of autoimmune and inflammatory diseases (12, 13, 14, 15, 16, 17, 18, 19). An important clue to the function of NK-T cells was provided by their interaction with professional APCs via CD1d (20, 21, 22). CD1d has some similarities in structure to MHC class 1 molecules (23), but it is not encoded by the MHC complex (the gene for CD1d is located on chromosome 1 in humans) and, in contrast to MHC class 1 molecules, is not polymorphic (20). While initially CD1d was believed to bind and present peptide Ags to T cells (24), more recent studies highlight its ability to present glycolipids and GPI-linked proteins (22, 25, 26, 27, 28, 29).
NK-T cells can become activated in a CD1d-restricted fashion with
subsequent proliferation and cytokine production, including IFN- and
IL-4. This CD1d-dependant activation can be enhanced by the addition of
specific glycolipid Ags, most notably the synthetic
-galactosylceramide (29, 30, 31, 32). The ligand recognized by
the NK-T cells in psoriasis and the functional consequences of such
interactions are unknown. Such glycolipid-reactive NK-T cells, besides
expressing CD94 and CD161, have a rather specific TCR in which an
invariant TCR -chain rearrangement (V24-JQ in humans,
V14-J 281 in mice) is paired with TCR-ß-chains, showing
predominance of only a few Vß segments (Vß11 in humans and Vß2,
-7, and -8 in mice) (29, 30, 31, 32). We previously isolated and
characterized one pathogenic CD1d-reactive T cell line bearing NKRs
CD94 and CD161 that did not bear the canonical V24-JQ TCR
rearrangement (8), but the precise relationship between
CD1d-reactive NK-T cells and T cells bearing NKRs is currently unclear.
Thus, in addition to documenting the presence of CD1d in psoriasis and
other skin disorders, we also searched for NK-T cells bearing V24
and Vß11 as well as CD94 and CD161.
Previous investigators focused on CD1d expression in the gastrointestinal tract, exploring its role in epithelial immunity and inflammatory bowel disease (33, 34, 35). This report documents patterns of CD1d expression by keratinocytes in vitro and in human skin and psoriasis in vivo. We also observed in psoriatic lesions the presence of intraepidermal lymphocytes expressing a variety of markers shared by classical NK-T cells. To explore the functional significance of these in vivo findings, NK-T cell clones were used to explore the potential for CD1d expressed on keratinocytes to trigger CD1d mediated immunologic responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three-millimeter punch biopsy specimens of normal adult human skin (NN skin; n = 16), symptomless skin from patients with psoriasis elsewhere (PN skin; n = 18), and active untreated psoriatic plaques (PP skin; n = 18) were obtained under local anesthesia after informed consent was given. Similarly, punch biopsy specimens were obtained from volunteers in which poison ivy Ag was applied epicutaneously in previously sensitized individuals (n = 3). In one patient with psoriasis, a biopsy specimen of PN skin 72 h after barrier function perturbation by repeated tape stripping was also examined. Thymic tissue was obtained from children undergoing elective cardiothoracic surgery. All tissue procurement was obtained with approval by institutional review boards. Tissue samples were snap-frozen in isopentane chilled in liquid nitrogen and stored at -80°C until cryostat sections (5 µm thick) were cut and placed on glass slides. Some of these biopsies were performed during the course of an earlier investigation (36).
Cell culture
Multipassaged normal human keratinocytes and an immortalized
human keratinocyte cell line (HaCaT) were grown in a low calcium,
serum-free medium (KGM, Clonetics, San Diego, CA) as previously
described (37, 38). Three different NK-T cell clones were
examined (DN2.D5, DN2.D6, and DN2.B9) that were generated as previously
described (29, 31). Briefly, a panel of double-negative
(DN) V24+ Vß11+ human
peripheral blood-derived NK-T cell clones was established by sequential
negative magnetic bead (Dynal, Lake Success, NY) isolation and positive
FACS sorting, followed by stimulation with PHA (Difco, Detroit, MI) and
IL-2 (70 U/ml) in the presence of irradiated (5000 rad) peripheral
blood mononuclear feeder cells. HeLa cells or CIR cells were
transfected with the expression vector pSR-Neo containing a cDNA
insert encoding CD1d as previously described (31).
Immunohistochemical staining
Cryostat sections were fixed using cold acetone followed by an avidin-biotin peroxidase staining procedure as previously described (39). Briefly, primary Abs (10 µg/ml) were incubated for 30 min at room temperature followed by subsequent steps according to the manufacturer’s instructions (Vectastain, Vector, Burlingame, CA). Positive detection was accomplished using 3-amino-4-ethylcarbazole as the chromagen producing a red reaction product with 1% hematoxylin as the counterstain (39).
Abs and cytokines
Primary murine Abs included anti-CD1d (clone NOR3.2 IgG,
BioSource, Camarillo, CA), anti-Vß11 (IgG, Becton Dickinson,
Mountain View, CA), and anti-V24; anti-CD161 and
anti-CD94 were purchased from Coulter (Hialeah, FL). Anti-ICAM-1
was purchased from Genzyme (Cambridge, MA). In addition, a panel of 15
different Abs raised against a soluble recombinant CD1d-IgG2a fusion
protein was employed including the following: CD1d8, CD1d19, CD1d27,
CD1d34, CD1d37, CD1d40, CD1d42, CD1d44, CD1d51, CD1d55, CD1d59, CD1d60,
CD1d68, CD1d69, and CD1d75. Several of the mAbs in this panel and the
methods used to produce them have been previously described
(40) For staining of cultured keratinocytes, eight-well
Lab-Tek slides (Nunc, Naperville, IL) were used. Semiconfluent
keratinocyte cultures were exposed to IFN-
(103 U/ml; Genzyme) for 48 h. The functional
blocking studies used anti-CD1d (IgM, CD1d59), anti-LFA-1 (IgG,
anti-CD18; Coulter), and pooled IgG/IgM (Coulter) as a control.
Proliferation studies and cytokine assay
NK-T cells (5 x 104/well) were
cultured in triplicate in RPMI and 10% FCS in 96-well round-bottom
tissue culture plates together with either keratinocytes or HeLa cells
(2.5 x 104/well). NK-T cell proliferation
was measured after 72 h by [3H]thymidine
incorporation (1 µCi/well) using target cells pretreated with
mitomycin C (0.1 mg/ml) for 1 h at 37°C (Sigma, St. Louis, MO)
as previously described (29). The synthetic glycolipid
(-galactosylceramide) used was provided by the Pharmaceutical
Research Laboratory/Kirin Brewery (Gunma, Japan) as previously
described (29, 31). To induce CD1d, keratinocytes were
pretreated with IFN- (103 U/ml) for 48 h,
washed, lifted with trypsin/EDTA followed by mitomycin treatment, and
washed three times by repeated centrifugation. No residual IFN- was
detected in these keratinocyte suspensions by ELISA. Stimulation of
NK-T cells was performed in the presence of 1 ng/ml
12-O-tetradecanoyl phorbol-13-acetate (TPA), as previously
described (29). Released cytokine levels for IFN- and
IL-4 were measured in duplicate after 48 h of coincubation between
NK-T cells with or without keratinocytes and HeLa cells by ELISA with
matched Ab pairs in relation to cytokine standards (R&D Systems,
Minneapolis, MN). In some experiments, Ficoll-Hypaque interface
mononuclear cells were stimulated using staphylococcal enterotoxins SEB
and SEC2 (1 µg/ml each; Toxin Technologies, Sarasota, FL) as
previously described (7).
Flow cytometry
Cells were incubated with the indicated primary and secondary Abs, washed, and resuspended in PBS containing 2% FCS. Cells were analyzed using a Coulter XL flow cytometer. The mAb CD1d 27 was used to detect CD1d, and anti-ICAM-1 was employed to compare with CD1d as well as isotype-matched control (Coulter). Data were analyzed with Elite software from Coulter.
RT-PCR
Detection of CD1d mRNA was performed using RT-PCR. Sequences of
the primers designed to specifically detect CD1d were: exon 2, sense,
5'-CTC CAG ATC TCG TCC TTC GCC AAT-3'; and exon 3, antisense, 5'-TTG
AAT GGC CAA GTT TAC CCA AAG-3'. These primers amplify an 
400-bp
fragment using an annealing temperature of 55°C and 35 cycles of PCR.
Control studies using a variety of different templates demonstrated the
reaction to be specific for CD1d, with no detectable amplification of
CD1a, -b, or -c transcription under these conditions (data not
shown).
Western blot and immunoprecipitation analysis
For Western blot analysis, lysates were performed on subconfluent cultures of keratinocytes and HaCaT cells (or CD1d-transfected CIR cells serving as a positive control) by incubating 75-cm2 flasks with 1 ml of lysis buffer (2% SDS, 5% 2-ME, 200 µM PMSF, and 65 mM Tris-HCl, pH 6.8) for 30 min at 4°C followed by harvesting with a cell scraper, sonication, and centrifugation at 100,000 x g for 30 min. Aliquots of the supernatant were applied to 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose. After blocking nonspecific protein binding using Tris-buffered saline, pH 7.6 (TBS)-milk (10 mM NaCl and 5% fat-free milk), the strips were incubated with mAb CD1d 75 overnight at 4°C, followed by a secondary Ab (goat anti-mouse Ig HRP, Dako, Carpinteria, CA) for 1 h at room temperature. After washing with TBS-Tween, binding of secondary Ab was revealed by chemiluminescence with the ECL kit (Amersham, Piscataway, NJ) on ECL film.
For immunoprecipitation/Western blot analysis HaCaT cells were grown in T-75 flasks, and lysates were prepared as described above. Lysates were centrifuged (1000 x g) to remove nuclei and debris and were precleared three times at 4°C with protein G-coupled Sepharose beads (Pharmacia). Equal volumes of precleared lysates were then incubated with 5% (v/v) protein G-coupled Sepharose beads to which nonbinding control (MPC-11; IgG2b) or CD1d-specific (CD1d51; IgG2b) mouse mAbs had been chemically cross-linked using dimethylsuberimidate as previously described (41). Forty-one lysates were incubated with mAb-coupled beads at 4°C for 6 h and then washed with TBS and split into two aliquots (i.e., with and without peptide N-glycosidase F (PNGase) F samples). Beads from each aliquot were suspended in 12 l of denaturation buffer (0.5% SDS and 1% 2-ME) and heated in a boiling water bath for 10 min, followed by addition of 2 l of 10% Nonidet P-40 and 2 l of 0.5 M sodium phosphate (pH 7.5 at 25°C). PNGase F (New England Biolabs, Beverly, MA) was then added to half the samples (500 U/sample) and incubated for 18 h at 37°C. Samples were then brought to 3 l with gel loading buffer (3% SDS, 0.02% bromophenol blue, 10% glycerol, and 62.5 mM Tris, pH 6.8) and electrophoresed on a 12% polyacrylamide-SDS slab gel. Western blotting to a polyvinylidene fluoride (NEN Life Science Products, Boston, MA) membrane was performed at 100 V for 1 h at 4°C. The membrane was blocked using 5% nonfat milk in TBS for 18 h, and then probed using mAb CD1d75 (5 µg/ml) in TBS with 0.1% Tween 20 and 0.05% NaN3 for 1 h at room temperature. The membrane was washed three times with TBS with 0.1% Tween 20 without NaN3 and developed using HRP-protein A and enhanced chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL) according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because previous studies suggested that CD1d is expressed in
thymus, we first assessed the staining pattern using a commercially
available anti-CD1d mAb (i.e., NOR3.2) on frozen sections of thymic
tissue. While CD1d staining was observed in cortical thymocytes,
dendritic cells, and endothelial cells with this mAb, a more striking
surface expression on Hassel’s corpuscles was noted, producing
concentric rings of positive immunoreactivity (Fig. 1
). This marked CD1d positivity of an
epithelial element of thymic tissue prompted an examination of the skin
for CD1d expression. Normal skin biopsy specimens were stained with NOR
3.2 mAb, which revealed CD1d to be consistently present in the three or
four outermost layers of keratinocytes in a distinct plasma membrane
staining pattern (Fig. 1B). Occasional staining of the basal
cell layer or midlayers of keratinocytes in the stratum spinosum was
also seen in some normal skin samples. Thus, most of the keratinocytes
in the stratum granulosum extending up to the lipid-rich stratum
corneum were CD1d positive. There was no apparent CD1d expression noted
on Langerhans cells, but weak, focal CD1d was present on dermal
dendritic cells and endothelial cells. A similar immunohistochemical
staining pattern, emphasizing CD1d expression by upper level
keratinocytes, was also present in prepsoriatic (PN) skin taken from
psoriatic patients (Fig. 1C). For both normal skin and PN
skin, CD1d was present focally on eccrine duct epithelium (Fig. 1C, inset) and acrosyringium (data not shown). The dermal
papillae and hair matrix were CD1d negative (Fig. 1D), but
the inner root sheath epithelium was diffusely positive, with some
anagen follicles displaying outer root sheath epithelium and sebocytes
being CD1d positive (Fig. 1E). No CD1d was discerned on
dermal mast cells or fibroblasts or in s.c. tissue sites (data not
shown).
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In contrast to normal skin and symptomless skin from psoriasis
patients, chronic psoriatic plaques had more extensive keratinocyte
CD1d expression (Fig. 1
, F and G). Because the
granular cell layer is generally absent in a psoriatic lesion
(42), CD1d expression began just beneath the large amount
of lipid-rich scale and extended to include 8–12 layers beneath the
stratum corneum to suprabasilar layers (Fig. 1G). CD1d was
primarily confined to plasma membrane, producing a broad
"chicken-wire" appearance of the plaques. There was focal CD1d
expression in the basal cell layer in several of the plaques, as well
as more diffuse and intense CD1d on dermal dendritic cells and
endothelial cells (Fig. 1, F and G). Because mild
trauma to the skin (i.e., repeated tape stripping) can trigger
psoriasis (43), we examined tissue samples for CD1d
expression. Within 72 h of barrier perturbation accomplished by
repeated tape stripping, symptomless (PN) skin acquired diffuse
epidermal CD1d expression in juxtaposition to the overlying
parakeratotic scale (Fig. 1H).
Because early lesions of psoriasis can resemble evolving allergic
contact dermatitis reactions (44) skin biopsies obtained
from sensitized individuals exposed epicutaneously to the hydrophobic
wax-containing oil of the poison ivy leaf (i.e., urushiol) were
examined (36). Eight hours after exposure, no changes in
CD1d expression were noted, but as early as 24 h following
exposure, biopsy samples revealed increased CD1d expression by
epidermal keratinocytes. At 48 h as mononuclear cells appeared in
the upper dermis and epidermis, keratinocytes in the mid and
superficial epidermal layers were CD1d positive (Fig. 1I).
To confirm and extend the pattern of CD1d expression in psoriatic
plaques as revealed by the NOR3.2 mAb, a panel of 15 different Abs
raised against CD1d was used for immunohistochemical analysis. Four of
these Abs did not produce any positive staining using cryostat sections
of psoriatic plaques (CD1d8, CD1d34, CD1d40, and CD1d60), but the other
Abs produced distinctive patterns that clustered into three groups. One
set of Abs (CD1d19 and CD1d37) against CD1d produced rather faint
staining of all cell layers of the epidermis (data not shown). The
second set of Abs (CD1d27 and CD1d42) produced a pattern most closely
resembling NOR3.2 staining, in that the basal cell layer had low to no
immunoreactivity, but there was strong and diffuse staining of all
suprabasilar keratinocytes of the plaque up to and including the
stratum corneum. In general, there was greater cytoplasmic reactivity
compared with NOR3.2 staining, but plasma membrane expression was
detectable with this set of anti-CD1d Abs (data not shown). The
third group of Abs (six different Abs; CD1d44, CD1d51, CD1d55, CD1d59,
CD1d68, CD1d69, and CD1d75) produced strong and diffuse staining of all
cell layers of the psoriatic epidermis (including the basal cell layer)
with cytoplasmic greater than plasma membrane intensity (data not
shown). To portray a representative profile of staining besides NOR3.2,
a different anti-CD1d mAb (CD1d27) is portrayed. Fig. 2 displays NN, PN, and PP skin reactivity
as well as thymic epithelium stained with CD1d27 mAb. Overall, the
staining results with this large panel of mAbs suggest that there may
be multiple epitopes on CD1d that are differentially expressed in
psoriatic plaques. The biochemical characterization of keratinocyte
CD1d expression in vitro and in vivo are further defined in a later
section.
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24, and Vß11 in normal skin and
psoriasis
Only rare CD161-, V24-, or Vß11-positive lymphocytes were
present in normal and symptomless (i.e., PN) skin (data not shown). A
positive control specimen was an invasive squamous cell carcinoma that
had prominent CD1d expression by the malignant cells accompanied by
numerous V24-positive lymphocytes (data not shown). Compared with
the sporadic presence of V24- or Vß11-positive lymphocytes in
normal skin, CD94- and CD161-positive T cells were more consistently
and more frequently found in skin biopsies of psoriatic plaques with
prominent CD1d expression. In the psoriatic plaques examined, a
striking abundance of CD161-positive T cells within CD1d-positive
keratinocyte layers was observed (Fig. 3, A–C). Beside CD161 expression by psoriatic lesional
intraepidermal T cells, CD94+ lymphocytes were
also present in the hyperplastic epidermis (Fig. 3D).
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Normal multipassaged human keratinocytes or HaCaT cells grown
using a low calcium, serum-free medium constitutively expressed CD1d at
a relatively low level with primarily cytoplasmic localization, as
revealed by immunohistochemical staining with either NOR3.2 (Fig. 4, upper panels) or CD1d27
mAbs (Fig. 4, lower panels). However, following 48 h of
exposure to IFN-, enhanced plasma membrane CD1d expression by
keratinocytes was observed (Fig. 4). Similarly, while proliferating
keratinocytes did not consistently express ICAM-1 (Fig. 4, upper
panels), after IFN- exposure plasma membrane ICAM-1 expression
was detected. Immortalized HaCaT cells (passages 134–135) produced
similar immunohistochemical staining profiles in response to IFN-
(data not shown).
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treatment, flow cytometry was performed (Fig. 4). While
the untreated keratinocytes had little or no surface CD1d expression or
ICAM-1 expression, exposure to IFN- produced marked expression of
both CD1d and ICAM-1, as measured by the peak shift for the mean
channel fluorescence.
To further characterize and extend these immunostaining results, RT-PCR
was performed as well as Western blot analysis. RT-PCR using RNA
extracted from untreated and IFN--treated normal keratinocytes and
HaCaT cells revealed the expected 400-bp product consistent with CD1d
mRNA transcripts, whereas omission of RT produced negative results
(data not shown). Western blot analysis revealed at least two distinct
immunoreactive bands with apparent Mr
of 45–47 kDa for both HaCaT cells and normal human keratinocytes as
well as in CD1d-transfected CIR cells that served as a positive control
(Fig. 5, upper panel). Western
blot analysis revealed that IFN- enhanced keratinocyte CD1d levels
(Fig. 5, upper right panel). Immunoprecipitation/Western
blot analysis of HaCaT cells revealed enhanced expression of a 47-kDa
form of CD1d by IFN- treatment (Fig. 5, lower panels).
The Mr of CD1d was reduced from 47 kDa
to two species of 32 and 30 kDa after PNGase F treatment in HaCaT cells
before and after exposure to IFN-. This enzymatic treatment
indicates the presence of glycosylated forms of CD1d. By Western blot
analysis the overexpression of CD1d in PP compared with NN skin
involved both 47- and 30-kDa species of CD1d (Fig. 5, lower right
panels).
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The ability of CD1d molecules expressed by keratinocytes to
stimulate human CD1d-restricted NK-T cell proliferation and IFN-
production (Fig. 6) was assessed using
previously described procedures (29) All three NK T cell
clones (i.e., DN2.D5, DN2.D6, and DN2.B9) express canonical TCR
-chain rearrangements as well as CD161, and variable levels of other
NK receptors, such as CD94 (data not shown). All these NK-T cell clones
responded with vigorous proliferation when mixed with equal numbers of
CD1d-transfected HeLa cells, but not mock-transfected HeLa cells. In a
typical experiment the NK-T cell clones alone incorporated
<103 cpm of
[3H]thymidine when cultured with
mock-transfected HeLa cells, which was increased 10- to 100-fold by
coculture with Cd1d-transfected HeLa cells plus TPA with or without the
glycolipid -galactosylceramide. For DN2.D5 and DN2.D6, only TPA
addition was necessary, while DN2.B9 cells required both TPA and the
synthetic CD1d-presented lipid Ag -galactosylceramide for optimal
proliferation with CD1d+ HeLa cells. None of
these NK-T cell clones significantly increased their
[3H]thymidine incorporation when combined with
either CD1d-negative or CD1d-positive keratinocytes (data not shown).
However, there was cluster formation noted in cultures in which the
NK-T cell clones were mixed with IFN--pretreated keratinocytes (to
induce CD1d), but not when untreated keratinocytes were added (Fig. 6).
This led us to explore the possibility that NK-T cells were being
activated to secrete cytokines rather than proliferate in response to
CD1d-positive keratinocytes. Initial experiments explored the relative
levels of IFN- and IL-4 produced by these three NK-T cell clones in
the absence of keratinocytes as well as when either untreated or
IFN--pretreated keratinocytes were added. Typically, the NK-T cell
clones spontaneously produced <5 pg/ml of IFN-, and when TPA/lipid
was added, this did not exceed 20 pg/ml. These IFN- levels were only
slightly increased (between 20 and 40 pg/ml of IFN-) when untreated
keratinocytes were added (Fig. 6). However, the NK-T cell clones
responded to the addition of IFN--pretreated (and extensively
washed) CD1d-positive keratinocytes in the presence of the TPA/lipid by
significantly enhancing IFN- production to >100 pg/ml for DN2-D6
and >400 pg/ml for DN2.B9 cells (Fig. 6). Culture wells containing
only IFN--pretreated and washed keratinocytes did not contain any
detectable IFN-, with a lower limit of detection of 3 pg/ml (data
not shown). Compared with the high induction of IFN- levels, none of
the three NK-T cell clones responded to either untreated or
IFN--pretreated keratinocytes by increasing their IL-4 levels (Fig. 6) by >25% above their constitutive levels in the absence of
keratinocytes (32 pg/ml of IL-4). While these same NK-T cells clones
can produce high levels of IL-4 following cross-linking of their TCRs
(29), combining the NK-T cell clones with keratinocytes
did not trigger similar levels of IL-4 production.
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when combined with CD1d-positive, but not CD1d-negative, HeLa
cells (Figs. 7 and 8). This reaction required the copresence
of suboptimal amounts of TPA (1 ng/ml) and -galactosylceramide (50
ng/ml). While no significant proliferation of DN2.B9 cells was induced
by either untreated or IFN--pretreated keratinocytes with or without
TPA and glycolipid, IFN- production by DN2.B9 cells was strongly
elevated with the CD1d-positive keratinocytes. As with the
CD1d-positive HeLa cells, the production of IFN- was enhanced by
addition of TPA and glycolipid. When IL-4 levels were measured in these
wells, the relatively low levels made constitutively by DN2.B9 cells
were not consistently or significantly increased when either
CD1d-positive HeLa cells or keratinocytes with or without IFN-
pretreatment were added (Fig. 6 and data not shown). It should be noted
that high levels of IFN- were produced when DN2.B9 cells were
combined with CD1d-transfected HeLa cells and TPA that did not require
any pretreatment with IFN-, further excluding the possible effects
of residual IFN- as contributing to the Th1 polarization or levels
of IFN- produced using keratinocytes as the stimulator cells.
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production by NK-T cells was blocked by anti-CD1d Ab
To determine the molecular basis for recognition by the DN2.B9
cells of target cells induced to express CD1d, various blocking Abs
were added (Figs. 7 and 8). A blocking Ab against CD1d as well as an
anti-LFA-1 Ab (but not control IgG/IgM Abs) significantly inhibited
the proliferation of DN2.B9 cells stimulated by CD1d-positive HeLa
cells. In addition, these same Abs reduced the production of IFN-
stimulated by CD1d-positive HeLa cells as well as CD1d-positive
keratinocytes. The anti-CD1d Ab did not inhibit SEB/SEC2 or
PHA-stimulated Ficoll-Hypaque mononuclear cell proliferation (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, but not IL-4. Previously, we
observed that cross-linking the TCR in these NK-T cell clones led to
enhancement of both IFN- and IL-4 levels (29),
suggesting that a selective stimulatory event was being mediated by
CD1d-expressing keratinocytes, leading to preferential IFN-
production. The failure to trigger significant NK-T cell proliferation
despite enhanced IFN- release has been previously observed with
other NK-T cell clones via recognition of class I MHC molecules
(45) The stimulation by CD1d of T cells bearing NK
receptors preferentially induces a cytokine switch to IFN-
(46, 47). Moreover, the differential induction of IFN-
production, but not IL-4, after the NK-T cell clones recognized CD1d on
keratinocytes has potentially important implications for psoriasis. Not
only is there overexpression of CD1d by psoriatic epidermal
keratinocytes and the presence of NK-T cells bearing CD94 and CD161,
but the cytokine IFN- has been shown to trigger psoriatic lesions
(2). We therefore postulate that a positive feedback loop
could be established in skin due to the presence of NK-T cells being
activated to produce IFN- upon contact with CD1d-positive
keratinocytes, leading to further CD1d expression and subsequent NK-T
cell release of more IFN-. The lack of a proliferative response by
NK-T cells to CD1d+ keratinocytes is also
consistent with the general number and distribution of CD94- and
CD161-positive NK-T cells in psoriasis. Thus, the NK-T cells are never
observed in tight clusters or in very large numbers as might be
expected if they were undergoing a local proliferative response;
rather, they are found as more evenly distributed single cells
throughout a psoriatic plaque.
In normal human skin CD1d was generally restricted to the outermost
keratinocyte layers in the stratum granulosum just beneath the
lipid-rich stratum corneum. It appeared that the plasma membrane
staining for CD1d was juxtaposed directly with the stratum corneum,
although CD1d could also be seen in some specimens to be present in the
basal cell layer. In addition to epidermal keratinocytes, CD1d was
detected on upper dermal dendritic cells, endothelium, eccrine ducts,
acrosyringium, and the pilosebaceous unit, except for the dermal
papillae and hair matrix cells. CD1d expression could be rapidly
modulated in vivo, as exemplified by the response of normal skin to
24 h of induction of allergic contact dermatitis involving the
poison ivy Ag or after repeated tape stripping of symptomless skin.
Similarly, addition of IFN- to cultured keratinocytes induced
strongly and diffuse expression of CD1d. Thus, like class I and class
II MHC Ags as well as ICAM-1 (48), keratinocytes can be
induced to express CD1d. A role for keratinocyte ICAM-1 expression in
this system was supported by the ability of the anti-LFA-1 mAb to
inhibit the NK-T cell proliferative and IFN- production
responses.
In psoriatic plaques CD1d expression was increased compared with that
in normal and symptomless skin, beginning in the suprabasilar layer and
extending to the outermost keratinocytes immediately beneath the
parakeratotic layer juxtaposed to the stratum corneum. CD161-positive T
cells were frequently observed in direct contact with keratinocytes
expressing CD1d in psoriatic plaques. Given this anatomical
juxtaposition, it is possible for various types of glycolipids in the
psoriatic scale to be directly exposed to the abundant keratinocyte
cell surface CD1d. Moreover, given the large hydrophobic binding
pockets in CD1d, the presence of CD1d on the outer layers of epidermis
in psoriatic plaques opens up the possibility that various glycolipids
present in the stratum corneum could play a role in triggering a
response by NK-T cells or other T cell subsets capable of recognizing
such glycolipids in the context of CD1d. During epidermal
differentiation keratinocytes produce different amounts and types of
various glycolipids, including glucosylceramides (49).
Alterations in these glycolipids in the stratum corneum can have a
significant impact on the barrier function of skin. Previously
investigators have focused on the direct proliferative effect of
glucosylceramides on epidermal keratinocytes (50).
However, it is also clear that barrier perturbation can initiate
cytokine cascades and thus influence inflammatory and mononuclear cell
activation (51, 52) The rapid and diffuse up-regulation of
CD1d expression seen in the symptomless skin following repeated tape
stripping demonstrates the highly inducible nature of CD1d expression
by keratinocytes. Furthermore, a reciprocal interaction appears likely,
because T cells can also influence barrier function (53).
Because cytokines such as IL-1 and TNF- are released after tape
stripping (52), these and other cytokines are being
studied to determine whether they can also induce CD1d expression by
keratinocytes. With regard to psoriasis, abnormalities in barrier
function have been well documented (54, 55), with both
quantitative and qualitative changes, including alterations in
glycolipids, that may not be similarly present in other forms of scaly
dermatitis such as atopic dermatitis (56). Thus, a cycle
can be envisioned in which pathogenic NK-T cells initiate barrier
abnormality, which, in turn, would generate glycolipids that could be
presented by keratinocyte CD1d and further activate
CD161+ T cells in psoriasis and allergic contact
dermatitis (57).
With regard to the triggering of NK-T cells, it is likely to be
important that these T cells bear both TCRs as well as NKRs. There is
precedent for synergistic interactions between these two classes of
receptors (58), and there is a possibility that such
synergistic activation of NK-T cells may have relevance to psoriasis.
The relative paucity of V24- or Vß11-positive lymphocytes in
psoriasis indicates that the T cells in psoriasis with NKRs may not
represent classical invariant TCR-bearing NK-T cells. Our recent
results characterizing a psoriatic pathogenic T cell line that was also
lacking a canonical TCR rearrangement but was CD1d reactive
(8) suggest greater heterogeneity of CD1d-reactive T cells
subsets beyond V24-positive NK-T cells (59). We
observed that when this NK-T cell line was injected into engrafted
autologous PN skin using a SCID mouse model, a psoriatic plaque was
created (8). This acute lesion was characterized by the
juxtaposition of NKR-bearing T cells in the epidermis containing
CD1d-positive keratinocytes (8). Taken together, these in
vivo findings along with the current in vitro findings support the idea
that NK-T cells may be playing an important pathophysiological role in
psoriasis (6, 7, 8, 60, 61).
Besides the ability of keratinocytes to initiate (48),
perpetuate (62), and terminate (63) immune
reactions involving conventional T cell responses to nominal Ags and
superantigens, CD1d expression may also imbue the keratinocyte with the
capacity to interact with NKR-bearing T cells. As a member of a
nonclassical, MHC-independent, Ag-presenting system, CD1d expression as
seen in psoriasis provides a novel opportunity for therapeutic
targeting and for understanding the immunologic and genetic basis of
psoriasis as well as the potential role for innate immunity in skin
disease (60, 61). At least one group has identified
familial linkage of psoriasis to chromosome 1 near the CD1d locus
(64). Furthermore, as suggested by the absence of CD1d in
the basal cell layer and its expression in suprabasalar keratinocytes
of psoriatic plaques, it appears that CD1d is influenced by the
differentiation status of the keratinocytes, much like other
neighboring genes on chromosome 1q21 also implicated in psoriasis
(65). It will be important to determine whether mutations
in CD1d are found in psoriatic patients, particularly at amino acid
residues that may influence recognition by T cells expressing NKRs
(66). In conclusion, CD1d is more widely distributed
throughout various body sites than originally observed, and
skin-derived keratinocytes expressing CD1d can be recognized by T cells
triggering IFN- production rapidly, which does not require preceding
T cell proliferation.
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Acknowledgments |
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Footnotes |
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2 B.B. and D.J. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Brian J. Nickoloff, Skin Cancer Research Laboratories, Loyola University Medical Center, Cardinal Bernardin Cancer Center, 2160 South First Avenue, Maywood, IL 60153.
4 Abbreviations used in this paper: NKR, NK cell receptor; NN skin, normal skin; PN skin, symptomless skin from patients with psoriasis elsewhere; PP skin, active untreated psoriatic plaques; DN, double negative; TPA, 12-O-tetradecanoyl phorbol-13-acetate; TBS, Tris-buffered saline, pH 7.6; PNGase F, peptide N-glycosidase F.
Received for publication May 5, 2000. Accepted for publication July 12, 2000.
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) and IL-4 (
) production when
three different NK-T cell clones were combined with either untreated
(none) or IFN-
