By
From the * Dermatology Branch, Epidermal Langerhans cells (LC) are immature dendritic cells (DC) located in close proximity
to the site of inoculation of infectious Leishmania major metacyclic promastigotes by sand flies. Using LC-like DC expanded from C57BL/6 fetal skin, we characterized interactions involving
several developmental stages of Leishmania and DC. We confirmed that L. major amastigotes,
but not promastigotes, efficiently entered LC-like DC. Parasite internalization was associated with activation manifested by upregulation of major histocompatibility complex (MHC) class I
and II surface antigens, increased expression of costimulatory molecules (CD40, CD54, CD80,
and CD86), and interleukin (IL)-12 p40 release within 18 h. L. major-induced IL-12 p70 release by DC required interferon evelopment of T cell-mediated immunity against foreign Ag necessitates prior Ag-nonspecific triggering
of APCs. With the recognition that dendritic cells (DC) are
uniquely able to initiate responses in naive T cells and that
DC also participate in Th cell education (for a review, see
references 1 and 2), considerable effort has been directed
towards identifying DC agonists and elucidating mechanisms that mediate DC activation. We have recently identified culture conditions that allow the expansion of epidermal Langerhans cell (LC)-like immature DC from murine
fetal skin (fetal skin-derived DC [FSDDC]; reference 3)
and have begun to characterize the response of FSDDC to
a variety of agonists (4, 5).
To study LC/DC-pathogen interactions and mechanisms responsible for pathogen-dependent DC activation,
we initiated experiments with FSDDC and Leishmania major. This experimental system was chosen because L. major
infection in mice is a well-established model for human cutaneous leishmaniasis (for a review, see reference 6), and
previous studies implicated LC as important participants in
the initiation phase of immune responses to Leishmania in
vivo (7). Prior in vitro studies of LC-L. major interactions have been hampered by technical difficulties associated with isolating keratinocyte-free LC and the spontaneous activation that results from removing LC from their
epidermal microenvironment.
In this study, we have taken advantage of the relatively
stable immature phenotype of FSDDC (4, 5) to assess the
DC-activating potential of the two developmental stages of
L. major that might interact with skin DC in the setting of
cutaneous leishmaniasis. We also evaluated cytokines produced by FSDDC and inflammatory macrophages (M Propagation of FSDDC.
Laboratory of Parasitic Diseases,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 20892
Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and prolonged (72 h) incubation. In contrast, infection of
inflammatory macrophages (M
) with amastigotes or promastigotes did not lead to significant
changes in surface antigen expression or cytokine production. These results suggest that skin
M
and DC are infected sequentially in cutaneous leishmaniasis and that they play distinct
roles in the inflammatory and immune response initiated by L. major. M
capture organisms
near the site of inoculation early in the course of infection after establishment of cellular immunity, and kill amastigotes but probably do not actively participate in T cell priming. In contrast,
skin DC are induced to express increased amounts of MHC antigens and costimulatory molecules and to release cytokines (including IL-12 p70) by exposure to L. major amastigotes that
ultimately accumulate in lesional tissue, and thus very likely initiate protective T helper cell type 1 immunity.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) in
response to L. major because cytokine-dependent Th education ultimately determines the outcome of infection (for a review, see references 6, and 10). We observed that
FSDDC preferentially ingested and were activated by L.
major amastigotes, and that FSDDC activation resulted in
IL-12 release. Although M
readily ingested amastigotes
(as well as promastigotes), they were not activated by infection. These data suggest that M
and DC are sequentially
parasitized in cutaneous leishmaniasis, and that skin DC,
rather than M
, are responsible for Th priming and the
initiation of Th education in this disease.
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Isolation of Inflammatory M.
Propagation and Isolation of L. major.
L. major clone V1 (MHOM/ IL/80/Friedlin) was cultured and different developmental stages were prepared as described (13). Infectious metacyclic promastigotes were isolated from stationary cultures by negative selection using peanut agglutinin. Amastigotes were prepared from homogenates of tissue derived from BALB/c footpad lesions via differential centrifugation and were used immediately or suspended in DMEM/10% fetal bovine serum (FBS)/7.5% DMSO and stored in liquid nitrogen. Isolated parasites were opsonized with 5% normal mouse serum before infection. Levels of LPS in parasite stock preparations were below the limit of detection (<0.1 endotoxin units/ml [LAL-test; BioWhittaker, Inc., Walkersville, MD]). Parasites were diluted 1:100 before use.Coculture Experiments with L. major and FSDDC or M.
Microscopy.
Morphologic changes in FSDDC aggregates in the coculture experiments were documented after 18 h using a video-linked inverted phase microscope (Eclipse TE 300; Nikon, Inc., Melville, NY). Parasite internalization was quantitated in DiffQuick-stained cytospin preparations using light microscopy. FSDDC aggregates were completely dissociated in calcium- and magnesium-free HBSS containing 1 mM EDTA (30 min at 37°C) before cytocentrifugation.Antibodies and Flow Cytometry.
Anti-CD16/CD32 (2.4G2) was provided by Julie Titus (National Cancer Institute, Bethesda). Anti-H-2Db (28-14-8), anti-I-Ab (2G9), anti-CD40 (3/23), anti-CD54 (3E2), anti-CD80 (1G10), and anti-CD86 (GL1) were purchased from PharMingen as biotin- or PE-modified mAbs. PE-streptavidin was obtained from Tago Inc. (Burlingame, CA). Cells were stained for surface Ag expression as described previously (3). Stained and paraformaldehyde (1% in PBS)-fixed cells were analyzed using a FACScan® flow cytometer equipped with CellQuest software (Becton Dickinson, Mountain View, CA).Quantitation of Cytokine Release.
Cytokine release into 18- and 72-h FSDDC and M ![]() |
Results |
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FSDDC were incubated with the different forms of serum-opsonized L. major, and cell-parasite interactions were studied by light microscopic examination of cytospin preparations. 18 h after amastigote addition, 36 ± 8% (n = 10) of the FSDDC were infected, and each infected cell contained up to six parasites (Fig. 1 A). In contrast, coincubation of FSDDC with promastigotes led to parasite attachment (Fig. 1 B), but only very low infection rates (7 ± 3%, n = 4).
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E-cadherin-mediated adhesion within aggregates of immature FSDDC can be viewed as a correlate of E-cadherin-mediated adhesion of LC to keratinocytes in epidermis (3, 14). Previous studies demonstrated that treatment of FSDDC with LPS or proinflammatory cytokines led to loss of adhesion within FSDDC aggregates and DC maturation (increased expression of MHC Ag and costimulatory molecules and enhanced APC activity [4]). Coincubation of FSDDC with L. major amastigotes for 18 h also induced dissociation of FSDDC aggregates into single, highly dendritic cells (Fig. 1 C). Although promastigotes adhered to FSDDC (Fig. 1 B), attachment was not accompanied by morphologic evidence of activation (Fig. 1 D).
Analysis of surface Ag expression using flow cytometry
confirmed that L. major amastigotes selectively induced
FSDDC activation and maturation. Amastigote infection of
FSDDC led to upregulation of MHC class I, class II, CD40,
CD54 (intracellular adhesion molecule 1 [ICAM-1]), CD80
(B7.1), and CD86 (B7.2) analogous to that observed after addition of the known DC activators LPS and IFN- (Fig. 2).
In contrast, coincubation of FSDDC with L. major promastigotes did not affect levels of the activation markers studied.
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Both infectious metacyclic promastigotes and tissue amastigotes of L. major are efficiently internalized by M (13). To determine if skin DC and M
were differentially activated
by exposure to Leishmania, the expression of relevant surface Ag by infected M
was quantitated. In the absence of
agonists, infiltrating tissue M
expressed low levels of
MHC class II, CD40, CD54, and CD86 (Fig. 3), as well as
MHC class I and CD80 (data not shown), consistent with
their derivation from nonimmune granulomas. Although
incubation of M
with L. major amastigotes or metacyclic
promastigotes resulted in frequent infection (infection rates
of 62 ± 12% [n = 4] and 53 ± 8% [n = 2], respectively)
within 18 h, neither life-cycle stage led to upregulation of
activation markers (Fig. 3).
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The outcome
of encounters between Ag-bearing APCs and naive T cells
depends, in part, on the nature of the cytokines released locally by APCs. This is particularly relevant in leishmaniasis because development of Th1-predominant protective immunity is clearly dependent on production of IL-12 (for a
review, see references 6 and 11). Thus, we identified and
quantitated cytokines released by Leishmania-infected FSDDC
and, for comparison, cytokines released by infected M.
Relative to untreated FSDDC, amastigote-infected FSDDC
released ~12-fold more IL-12 p40 (P < 0.05, n = 9) and
increased amounts of TNF-
(P < 0.05, n = 9) into supernatants (see Table 1), whereas incubation of FSDDC with
promastigotes did not stimulate cytokine production. Small
amounts of IL-1
, IL-1
, and IL-6 were also released by
FSDDC (Table 1, and data not shown), but production of
these cytokines was not augmented by infection with
amastigotes. IL-4, IL-10, IL-12 p70, and IFN-
were not
detected in 18-h FSDDC supernatants (data not shown).
Interestingly, prior infection of FSDDC with amastigotes
did not inhibit IL-12 release induced by relatively high
concentrations of LPS and IFN-
(Table 1). Consistent
with previous reports (15), bioactive IL-12 (p70) was detected only in 72-h supernatants of FSDDC (Table 2).
Amastigote-induced FSDDC IL-12 p70 release also required addition of IFN-
. Note that similar amounts of
IL-12 p70 were released by amastigotes plus IFN-
and
maximally stimulated LPS plus IFN-
plus anti-CD40- treated FSDDC.
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These results contrast with those obtained with M. Infection of nonimmune tissue M
with L. major did not induce significantly increased production of IL-12 or other
cytokines (Table 1). In addition, LPS plus IFN-
-induced
IL-12 release by M
was inhibited by infection with
amastigotes before stimulation. These findings confirm previous observations made with M
infected with metacyclic promastigotes or amastigotes (6, 13, 16, 17), and highlight the differential effects of Leishmania on DC and M
.
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Discussion |
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Cutaneous leishmaniasis is initiated by inoculation of
small numbers of L. major metacyclic promastigotes into the
dermis (for a review, see references 6 and 18). Although
M ingest promastigotes, they are not activated and are
rendered selectively unable to produce the Th1-promoting
cytokine IL-12 in response to inflammatory mediators (references 6, 13, and 16, and this study). In addition, the evidence that Leishmania-infected M
migrate to regional
lymph nodes and trigger responses in naive T cells is not
compelling (8). Thus, although dermal M
represent the
initial site of parasite proliferation after epicutaneous inoculation and IFN-
-activated M
are ultimately responsible for the destruction of organisms (6, 18), M
may not play a
role in the induction of anti-Leishmania immunity.
The results of this and previous studies suggest that DC
rather than M are responsible for T cell priming in leishmaniasis. Because the L. major life form that is inoculated into
skin does not parasitize immature DC (references 19 and 20,
and this study), it seems likely that LC/DC infection occurs
subsequent to amastigote release by M
. Exposure of FSDDC to amastigotes led to upregulation of MHC and costimulatory molecules and loss of E-cadherin-mediated adhesion within aggregates. The former observation is consistent
with earlier results which indicated that amastigotes induced a
transient increase in MHC class II biosynthesis in LC (21).
The latter finding demonstrates that L. major amastigote-
induced activation of FSDDC is associated with downregulation of E-cadherin expression and/or function as might be
expected before mobilization of LC from epidermis to lymph
nodes (4, 22). Although coincubation with amastigotes did
not lead to infection of the entire FSDDC population, MHC and costimulatory molecules were upregulated on almost all
cells. This may reflect a bystander effect (e.g., activation of
uninfected DC by TNF-
release by infected cells [3]) or
effects of organism fragments/subcellular fractions of parasites
that are not visualized by light microscopy.
IL-12 plays an important role in the immunophysiology
of experimental leishmaniasis and is required for the development of protective Th1-predominant immunity (for a
review, see reference 6). In addition to directly facilitating
Th1 education, IL-12 activates NK cells to become effectors and produce IFN- (23), which may also promote
Th1 development and/or augment M
leishmaniacidal activity. Because Leishmania-infected tissue M
do not release IL-12 spontaneously or in response to potent stimuli
(references 6, 13, 16, and 17, and this study), it is unlikely
that M
are the primary source of IL-12 in lesional tissue.
Recent studies indicate that systemic administration of
Toxoplasma gondii extracts (24) or Leishmania donovani
amastigotes (25) results in rapid IL-12 p40 accumulation in
DC in lymphoid tissue, but not in M. These observations
localize IL-12 production to the APCs and the microenvironment that are thought to be critical for T cell priming.
Our results indicate that parasitized DC are a likely source
of IL-12 in leishmaniasis, and demonstrate that amastigotes
directly stimulate IL-12 production by skin DC. In addition to inducing IL-12 release, our data also indicate that L.
major amastigotes induce DC maturation. We hypothesize
that local activation of skin DC by Leishmania, mobilization
of skin DC, and localization of Ag-bearing, IL-12-producing mature DC in regional lymph nodes is required for development of protective immunity. The delayed appearance of IL-12 p40 transcripts in lymph nodes draining
murine skin inoculated with L. major (6) has been attributed to dissemination of amastigotes from skin to lymph nodes followed by IL-12 synthesis within M
. An alternative interpretation is that parasites are conveyed to lymph
nodes by skin-derived DC, and that IL-12 is produced
within infected DC. The scenario we propose is also consistent with previous data indicating that infected DC can be
recovered from lymph nodes that drain murine skin inoculated with L. major, and that L. major-infected LC can initiate primary anti-Leishmania responses in T cells after injection into naive mice whereas infected M
cannot (8, 9, 26).
We envision that skin DC play a particularly important
role as transporters of parasites/Ag to lymph nodes in cutaneous leishmaniasis initiated by small numbers of parasites
(e.g., numbers of parasites comparable to those introduced
by sand flies), where extracutaneous dissemination occurs
only after significant proliferation of Leishmania in the dermis. The observation that development of protective immunity in naturally acquired infections in people (27) and in C57BL/6 mice inoculated with small numbers of metacyclic promastigotes (D.L. Sacks, unpublished observations)
is delayed relative to that which occurs after administration
of standard inocula suggests the existence of a threshold requirement that must be satisfied before priming of naive T
cells can occur. One possibility is that recruitment of epidermal LC or dermal DC into sites of inoculation requires
elaboration of proinflammatory cytokines (e.g., IL-1 or
TNF-) or chemokines (16, 28), which are released only after a significant parasite load accumulates. Alternatively
(or in addition), priming may require that relatively large
numbers of extracellular amastigotes are available to activate resident skin DC. The latter possibility would suggest
that parasites that are effectively sequestered within M
in
the early stages of infection would not initiate priming and
also might not lead to a dramatic inflammatory response.
Additional experiments will be required to distinguish between these, and other, potential explanations.
Delineation of the role that skin DC play in T cell priming in cutaneous leishmaniasis is important for several reasons. First, leishmaniasis is a significant world health problem
for which no effective vaccine exists. Development of a useful vaccine will require identification of adjuvants that elicit
protective responses as well as relevant Ag. Because DC are
likely to be involved in Th1 education, DC agonists may be
potent adjuvants. Second, it is interesting that DC and M
are differentially activated by L. major. This suggests that
Leishmania may initiate immune responses via mechanisms
that are distinct from those activated by other parasites (e.g.,
the potent M
activators T. gondii (29) and Listeria monocytogenes [30]). Elucidation of these mechanisms will further our
understanding of the roles DC and M
play in the pathophysiology of various parasitic diseases and may also improve
our ability to prevent or treat these common infections.
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Footnotes |
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Address correspondence to Mark C. Udey, Dermatology Branch, National Cancer Institute, National Institutes of Health, Bldg. 10, Rm. 12N238, 9000 Rockville Pike, Bethesda, MD 20892-1908.
Received for publication 11 June 1998 and in revised form 12 August 1998.
E. von Stebut was supported by the Deutsche Forschungsgemeinschaft (Ste 833/2-1), and Y. Belkaid and T. Jakob were funded through the Fogarty International Center, National Institutes of Health.The authors thank Bai Nguyen and Mark Wilson for expert technical assistance, Harry Schaefer for preparing the figures, and Drs. Patricia Walker and Jonathan Vogel for helpful discussions.
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References |
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1. | Steinman, R.M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9: 271-296 [Medline]. |
2. | Banchereau, J., and R.M. Steinman. 1998. Dendritic cells and the control of immunity. Nature. 392: 245-252 [Medline]. |
3. | Jakob, T., A. Saitoh, and M.C. Udey. 1997. E-cadherin- mediated adhesion involving Langerhans cell-like dendritic cells expanded from murine fetal skin. J. Immunol. 159: 2693-2701 [Abstract]. |
4. |
Jakob, T., and
M.C. Udey.
1998.
Regulation of E-cadherin-mediated adhesion in Langerhans cell-like dendritic cells by
inflammatory mediators that mobilize Langerhans cells in
vivo.
J. Immunol.
160:
4067-4073
|
5. | Jakob, T., P. Walker, A. Krieg, M. Udey, and J. Vogel. 1998. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides. A role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J. Immunol. In press. |
6. | Reiner, S.L., and R.M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13: 151-177 [Medline]. |
7. | Will, A., C. Blank, M. Rollinghoff, and H. Moll. 1992. Murine epidermal Langerhans cells are potent stimulators of an antigen-specific T cell response to Leishmania major, the cause of cutaneous leishmaniasis. Eur. J. Immunol. 22: 1341-1347 [Medline]. |
8. | Moll, H., H. Fuchs, C. Blank, and M. Rollinghoff. 1993. Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Eur. J. Immunol. 23: 1595-1601 [Medline]. |
9. | Moll, H. 1995. The Immune Functions of Epidermal Langerhans Cells. R.G. Landes Company, Austin, TX. 190 pp. |
10. | Launois, P., J. Louis, and G. Milon. 1997. The fate and persistence of Leishmania major in mice of different genetic backgrounds: an example of exploitation of the immune system by intracellular parasites. Parasitology. 115: S25-S32 [Medline]. |
11. | O'Garra, A.. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity. 8: 275-283 [Medline]. |
12. | Gorham, J.D., M.L. Gueler, and K.M. Murphy. 1997. Genetic control of interleukin 12 responsiveness: implications for disease pathogenesis. J. Mol. Med. 75: 502-511 [Medline]. |
13. | Belkaid, Y., B. Butcher, and D.L. Sacks. 1998. Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania-infected cells. Eur. J. Immunol. 28: 1389-1400 [Medline]. |
14. | Tang, A., M. Amagai, L.G. Granger, J.R. Stanley, and M.C. Udey. 1993. Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature. 361: 82-85 [Medline]. |
15. | Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, and G. Schuler. 1996. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J. Exp. Med. 184: 741-746 [Abstract]. |
16. | Carrera, L., R.T. Gazzinelli, R. Badolato, S. Henry, W. Muller, R. Kuhn, and D.L. Sacks. 1996. Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. J. Exp. Med. 183: 515-526 [Abstract]. |
17. | Sartori, A., M. Oliveira, P. Scott, and G. Trinchieri. 1997. Metacyclogenesis modulates the ability of Leishmania promastigotes to induce IL-12 production in human mononuclear cells. J. Immunol. 159: 2849-2857 [Abstract]. |
18. | Bogdan, C., and M. Rollinghoff. 1998. The immune response to Leishmania: mechanisms of parasite control and evasion. Int. J. Parasitol. 28: 121-134 [Medline]. |
19. | Locksley, R.M., F.P. Heinzel, J.E. Fankhauser, C.S. Nelson, and M.D. Sadick. 1988. Cutaneous host defense in leishmaniasis: interaction of isolated dermal macrophages and epidermal Langerhans cells with the insect-stage promastigote. Infect. Immun. 56: 336-342 [Medline]. |
20. | Blank, C., H. Fuchs, K. Rappersberger, M. Rollinghoff, and H. Moll. 1993. Parasitism of epidermal Langerhans cells in experimental cutaneous leishmaniasis with Leishmania major. J. Infect. Dis. 167: 418-425 [Medline]. |
21. | Flohe, S., T. Lang, and H. Moll. 1997. Synthesis, stability, and subcellular distribution of major histocompatibility complex class II molecules in Langerhans cells infected with Leishmania major. Infect. Immun. 65: 3444-3450 [Abstract]. |
22. | Schwarzenberger, K., and M.C. Udey. 1996. Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cell E-cadherin expression in situ. J. Investig. Dermatol. 106: 553-558 [Abstract]. |
23. | Trinchieri, G.. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13: 251-276 [Medline]. |
24. |
Reis e Sousa, C.,
S. Hieny,
T. Scharton-Kersten,
D. Jankovic,
H. Charest,
R.N. Germain, and
A. Sher.
1997.
In vivo microbial stimulation induces rapid CD40 ligand-independent
production of interleukin 12 by dendritic cells and their redistribution to T cell areas.
J. Exp. Med.
186:
1819-1829
|
25. | Gorak, P.M.A., C.R. Engwerda, and P.M. Kaye. 1998. Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection. Eur. J. Immunol. 28: 687-695 [Medline]. |
26. | Moll, H., S. Flohe, and M. Rollinghoff. 1995. Dendritic cells in Leishmania major-immune mice harbor persistent parasites and mediate an antigen-specific T cell immune response. Eur. J. Immunol. 25: 693-699 [Medline]. |
27. | Melby, P.C.. 1991. Experimental leishmaniasis in humans: review. Rev. Infect. Dis. 13: 1009-1017 [Medline]. |
28. | Racoosin, E., and S. Beverley. 1997. Leishmania major: promastigotes induce expression of a subset of chemokine genes in murine macrophages. Exp. Parasitol. 85: 283-295 [Medline]. |
29. |
Gazzinelli, R.T.,
S. Hieny,
T.A. Wynn,
S. Wolf, and
A. Sher.
1993.
Interleukin 12 is required for the T-lymphocyte-independent induction of interferon ![]() |
30. |
Tripp, C.S.,
S.F. Wolf, and
E.R. Unanue.
1993.
Interleukin
12 and tumor necrosis factor ![]() ![]() |