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Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-10. These data suggest that L3 exert a down-regulatory response in epidermal LC that leads to a diminished capacity of these cells to activate CD4+ T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LC are bone marrow-derived cells that are present in all epithelial tissues (1) and are essential for the initiation and propagation of immune responses against foreign Ag in the skin. Before contact with Ag, LC express low levels of MHC class I and II and costimulatory molecules and are poor stimulators of unprimed T cells. Upon contact with Ag, these cells become activated and migrate to the regional lymph node, where they act as mature APC (2, 3). It has been suggested that TNF-
and IL-1
are the two independent cytokine signals required for migration of LC. Both are up-regulated following various forms of skin trauma and result in necessary physiologic changes to allow for migration from the skin to the draining lymph nodes (4). LC produce a variety of mediators, including cytokines such as IL-1, IL-6, IL-12, and IL-18 that are capable of playing a role in the initiation and modulation of immune responses in the skin (5).
Several studies using animal models have addressed the interaction of LC with parasites and the consequences of these interactions. It has been demonstrated that LC loaded with Leishmania major Ag administered i.v. are highly efficient in inducing protective immunity against cutaneous leishmaniasis. Indeed, these LC migrate to the spleen, where they first cause a primary cytokine response; over weeks, they induce a characteristic shift toward development of Th1 cells mediated by LC-derived IL-12 (6). In addition, uptake of L. major amastigotes by skin-derived dendritic cells (DC) results in activation and IL-12 release-derived DC (7). In murine infection with schistosomula, LC were shown to become activated, although their migration to the lymph nodes was impaired; this inhibitory effect was shown to be mediated by excreted/secreted lipophilic factors produced by parasite larvae, particularly by PGD2 (8). Other investigators have shown that precutaneous exposure of guinea pigs to attenuated or normal larvae of Schistosoma mansoni results in marked change of distribution and morphology of epidermal LC (9).
Despite the demonstration of hyperproliferation of KC in the skin of filaria-infected patients with elephantiasis and/or with long-standing severe lymphatic obstruction (10), there have been no studies addressing the earliest interaction between L3 and the human host. Previously, we used the bloodborne MF stage of Brugia malayi (Bm) (11, 12) to study the interaction between this stage of the parasite and monocyte-derived DC, although this interaction occurs in the context of long-standing, chronic infection. We have shown that both MF Ag and live MF impair the function of monocyte-derived DC to produce IL-12 and IL-10 and to induce cytokines by CD4+ T cells (11, 12). Furthermore, using microarray analysis, we have shown that, whereas inflammatory responses in monocyte-derived macrophages were induced by live L3 of Bm, monocyte-derived DC were not affected by this parasite (13). One possible explanation is that, because of its route of entry, L3 may interact with different types of DC such as LC. Therefore, how the infective stage of this parasite initiates or evades the initial host response is of paramount importance not only for understanding parasite survival but also for gaining insight into CD4+ T cell subset differentiation following helminth infection.
Considering the role played by LC both in the initiation of the immune response and in presentation of parasite Ag in the regional lymph node, we have examined the consequences of the interaction between the L3 stage of filarial parasites and LC through a system that relies on human epithelium-derived LC obtained in a physiologically native state (14). Using human epithelial explants exposed to parasite L3, we have demonstrated that interaction of L3 with these explants results in migration of viable LC from the explants that have markedly diminished expression of MHC class I and II. By microarray and real-time RT-PCR, we can also demonstrate L3-induced diminished expression of IL-8 and a multitude of genes involved in Ag presentation that translated into a reduced capacity to stimulate MLR or to activate autologous CD4+ T cell or produce cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As described previously (14), skin blisters (1 cm in diameter) were raised from healthy human volunteers by vacuum suction and heat. Once the blisters were formed, the epidermal sheets were surgically removed and washed consecutively in three petri dishes containing sterile PBS (Biofluids, Rockville, MD). This procedure was done under a protocol approved by the Institutional Review Board (National Institute of Allergy and Infectious Diseases, National Institutes of Health).
Explant culture conditions
Epidermal tissue explants were cultured in 24-well plates (Costar, Cambridge, MA), one explant per well, in 0.5 ml of complete RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 20 mM glutamine (BioWhittaker), 10% heat-inactivated human FCS (Harlan Bioproducts for Science, Madison, WI), 100 IU/ml penicillin, and 100 g/ml streptomycin (Biosource International, Rockville, MD). Explants from each donor were cultured in medium with or without five live L3 per explant for 72 h. Crawl-out LC were harvested at 72 h, counted, and used. In some experiments, the L3 were separated from the explants by 3-µm Transwells (Costar).
Parasite preparation
Bm live L3 isolated from whole infected mosquitoes (Aedes aegypti) were provided by Dr. J. McCall (University of Georgia, Athens, GA). L3 were then incubated in a six-well plate in RPMI 1640 plus penicillin/streptomycin/amphotericin B, 200 mM L-glutamine, and gentamicin for 1 h, following which the L3 were isolated individually, washed twice in the same medium, counted, and replated in a six-well plate before incubation with skin explants. Schistosomula of S. mansoni were provided by the laboratory of Dr. A. Sher (National Institute of Allergy and Infectious Diseases, National Institutes of Health).
Flow cytometry
Staining of cells with Ab was conducted according to standard protocols. Propidium iodide (Sigma-Aldrich, St. Louis, MO) was used to exclude nonviable cells from the analysis. LC were harvested and washed with FACS medium (HBSS without phenol red and without Ca2+/Mg2+ (BioWhittaker)) containing 0.2% human serum albumin (Sigma-Aldrich) and 0.2% sodium azide (Sigma-Aldrich). Cells were incubated with human gammaglobulin (Sigma-Aldrich) at 10 mg/ml for 10 min at 4°C to inhibit nonspecific binding of mAb through FcR. Cells were then incubated with specific mAb conjugated with FITC or PE at saturating concentrations for 30 min at 4°C, washed twice with FACS medium, and analyzed using a FACSCalibur (BD Biosciences, San José, CA) and CellQuest software (BD Biosciences). All Ab used were mouse anti-human mAb and consisted of the following: CD1a-PE (clone VIT6B; Caltag, Burlingame, CA); CD40-FITC (clone 14G7; Caltag); CD80 (B7-1)-FITC (clone L307.4; BD PharMingen, San Diego, CA); CD86 (B7-2)-FITC (clone 2331; BD PharMingen); CD83-PE (clone HB15e; BD PharMingen); HLA-A, B,C-FITC (clone G46-2.6; BD PharMingen); HLA-DR-FITC (clone L243; BD PharMingen); CCR5-PE (clone 2D7; BD PharMingen); CCR6-PE (clone 11A9; BD PharMingen); and CCR7-PE (clone 150503; R&D Systems, Minneapolis, MN).
Cytokine assays
All cytokines were detected in culture supernatants using cytokine-specific ELISA. For IL-12p70, paired Ab from R&D Systems, and for IL-12p40, IL-5, IFN-, and IL-10, paired Ab from BD PharMingen, were used. Assays were performed according to the manufacturer’s guidelines. The lower limits of detection for the assays were as follows: for IL-12p70, 33 pg/ml; for IL-12p40, 78 pg/ml; for IFN- and for IL-10, 39 pg/ml; and for IL-5, 19 pg/ml. IL-8 and IL-18 detection was performed by Pierce Biotechnology (Boston, MA) using Searchlight proteome arrays.
RNA preparation
Explants were cultured in medium alone or were exposed to live L3 for 6, 24, 48, or 72 h, after which the cells were harvested and total RNA was prepared using the RNAeasy mini-kit (Qiagen, Valencia, CA). RNA from cells obtained from four independent donors were pooled to make cDNA for real-time RT-PCR and cRNA for microarray analysis.
Microarray analysis
Total RNA was used to generate cRNA probes. Preparation of cRNA, hybridization, and scanning of the HU95 arrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Briefly, 12–15 µg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice; Life Technologies, Gaithersburg, MD) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of poly(T) (Genset Oligos, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with the Bioarray HighYield RNA transcription labeling kit (Enzo, Farmingdale, NY). The labeled cRNA was purified using RNAeasy spin columns (Qiagen). The labeled cRNA samples were fragmented at 94°C before hybridization. Labeled cRNA was hybridized to the HU95A microarray while rotating at 60 rpm for 
16 h at 45°C. After hybridization, the microarray was washed using the Affymetrix Fluidics Station in buffer containing biotinylated anti-streptavidin Ab (Vector Laboratories, Burlingame, CA) for 10 min at 25°C and stained with streptavidin PE (final concentration, 10 µg/ml; Molecular Probes, Eugene, OR) for 10 min at 25°C. Subsequently, the microarray was washed, restained with streptavidin PE (10 min, 25°C), and washed again before measuring the fluorescence bound to the microarray at 570 nm in an Affymetrix scanner.
Microarray data processing
Images of scanned Affymetrix GeneChips were processed using the software and parameter settings suggested by the manufacturer (Affymetrix). The processed fluorescence values were placed into an Excel table (Microsoft Excel Analysis Tools; Microsoft, Seattle, WA) in which rows represented genes and columns represented experimental conditions. We performed all data filtering on a Microsoft Excel spreadsheet. We chose a filtering strategy based on present call and fold change of 
3 for up-regulation and 
3 for down-regulation of the gene. Under all conditions, L3-exposed values were compared with those of unexposed DC at the given time point. When filtering was complete, the results were clustered using a Web-based hierarchical clustering program (15). For categorizing genes, a Web-based program (www.libgenechip.niaid.nih.gov; Laboratory of Immunopathogenesis and Bioinformatics, National Institute of Allergy and Infectious Diseases, National Institutes of Health) was used.
Real-time RT-PCR (TaqMan)
Predeveloped TaqMan assay reagents (Applied Biosystems, Foster City, CA) were used to detect IL-8, IL-18, and HLA-DB1. Probes, reagents, and equipment were used as recommended by the manufacturer (Applied Biosystems).
Immunofluorescent staining of LC
LC were harvested 72 h after culture. Coverslips were coated with poly-L-lysine diluted 1/10 in PBS for 1 h at 37°C and then washed with PBS. Cells were incubated with human gammaglobulin (Sigma-Aldrich) at 10 mg/ml for 10 min at 4°C to prevent nonspecific binding of mAb to FcR and were fixed with 4% PFA for 1 h at room temperature. Following a wash with PBS, they were resuspended in a small volume of 60 µl, placed on the coverslips, and stained with polyclonal rabbit anti-L3 Ab or control rabbit sera for 1 h. After washing, the secondary Ab, Alexa conjugated (Molecular Probes), was used to detect rabbit anti-L3 Ab. Immunofluorescent microscopy was then used.
Isolation of T cells
Blood (60 ml) was obtained from the same normal volunteers that provided the skin blisters. For the MLR studies, blood was obtained from normal blood donors (National Institutes of Health) by apheresis. Resting CD4+ T cells were obtained from PBMC by negative selection using a mixture of mAb and rigorous immunomagnetic negative selection with BioMag beads (Polysciences, Warrington, PA) bound to goat anti-mouse IgG (H+L) as described previously (16). Purity of the isolated cells was shown by flow cytometry to be >96%. The selected CD4+ T cells were free of monocytes based both on flow cytometry and on the criterion that there was no proliferative response to optimal concentrations (1/200 dilution) of PHA (M form) (Life Technologies).
In vitro CD4+ T cell activation
Unexposed or L3-exposed LC were harvested 72 h postculture and cocultured with autologous CD4+ T cells (at a 1:50 LC:T cell ratio based on the limited number of LC obtained) either in medium or 10 µg/ml solubilized anti-CD3 in 48-well tissue culture plates (Costar). Thymidine incorporation was measured on day 7 after a 24-h pulse with [3H]thymidine solution (5 mCi/ml; 2 mCi/mmol specific activity; New England Nuclear, Beverly, MA). Incorporation of radioactive label was measured using liquid scintillation spectroscopy. Results are expressed as the arithmetic mean cpm of triplicate cultures. In parallel experiments, supernatants were collected at 2 days for cytokine measurement. For cytokine measurement, anti-CD3 was immobilized on plate at 1 µg/ml and then washed. CD4+ T cells cocultured with unexposed or L3-exposed LC at a 1:50 LC:T cell ratio. Supernatants were collected 48 h after activation.
Mixed leukocyte reaction
Purified CD4+ T cells (50,000) were cultured in 96-well U-bottom microplates with 1000 unexposed or L3-exposed LC. Thymidine incorporation was measured on day 7 after a 24-h pulse with [3H]thymidine solution (5 mCi/ml; 2 mCi/mmol specific activity; New England Nuclear). Incorporation of radioactive label was measured using liquid scintillation spectroscopy. Results are expressed as the arithmetic mean cpm of triplicate cultures.
Statistical analysis
The nonparametric Wilcoxon signed rank test was used throughout. All statistical analyses were performed with StatView 5 (SAS Institute, Cary, NC).
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Results |
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To mimic the exposure of human skin to live Bm L3, we obtained epithelial tissue explants containing immature LC by suction blistering of healthy human skin (Fig. 1, a and b). The blister suction procedure splits the skin through the lamina lucida, leaving the blister roof (epidermis) intact. The explants were then placed in a 24-well culture plate, and LC spontaneously migrated from the epithelial tissue into the tissue culture medium over a 24- to 72-h period. After 72 h of culture, the LC that crawled out were collected, enumerated, and used for confirmation of their morphology. LC showed a typical morphology, expressing high levels of CD1a, CD80, CD86, CD40, MHC class I and II, and CD83, but no expression of CCR5 and low expression of CCR6 (data not shown). To mimic the physiologic conditions of Bm infection, some of the epithelial explants were cultured with five live L3 per one tissue explant (1 cm in diameter). Following exposure, at a time at which the number of LC that migrated out of the explants was maximal (72 h), the cells were harvested and cytospins were prepared (Fig. 1c). Immunofluorescent microscopy following staining with polyclonal anti-L3 Ab demonstrates that the LC very clearly take up and internalize Ag from the (E/S) products of live L3 (Fig. 1d).
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From one epithelial tissue explant (1 cm in diameter), between 1.2 and 10 x 104 LC (representing 60–80% of the total cells, based on the expression of CD1a) could be collected. Exposure of these explants to live L3 for 72 h (Fig. 2a) or 48 h (data not shown) did not inhibit the migration of LC from the explants; in virtually all donors, the number of migrating LC after exposure to live L3 significantly exceeded that of those without L3 (1,800–25,000 cells/blister compared with 1,200–12,000, respectively; p = 0.0012) (Fig. 2a). Furthermore, live L3 did not inhibit the cell viability of LC as shown both by trypan blue exclusion (Fig. 2a) and by propidium iodine staining (b). In five donors, when the L3 were physically separated from the explants by 3-µm Transwells, there was no difference in cell viability (trypan blue or propidium iodine (data not shown)) from either L3-exposed or -unexposed explants; however, in all donors, the number of migrating cells slightly diminished under conditions in which the Transwells were used (Fig. 2a, inset).
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To investigate whether exposure to L3 and subsequent internalization of L3 Ag by LC would result in changes in the expression of cell surface molecules, LC cultured with or without live L3 were analyzed by flow cytometry. Live L3 clearly down-regulated the expression of MHC class I and class II (Fig. 3) without changing the surface expression of other molecules such as CD1a, CD40, CD80, CD86, CCR5, and CCR6 (data not shown). It should be noted that unactivated KC do not have a significant surface expression of MHC class II, so that the down-regulation of MHC class II molecules by L3 appears to be LC specific.
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Having demonstrated that live L3 do not interfere with the viability or migration of LC but down-regulate MHC class I and II expression, we assessed more globally the effect of live L3 on epithelial explant cells using microarray analysis and RNA prepared from unexposed skin explants and those exposed to five live L3 per blister at four time points (6, 24, 48, or 72 h) (Fig. 4). Using hierarchical clustering to compare the L3-exposed explants to the unexposed ones at different time points, it can be seen that the majority of changes in gene expression profiles were observed after 72 h of exposure to L3. As shown in Fig. 4, the modification at 72 h was not due to the changes in basal expression level of these genes at 72 h. Of 12,000 genes expressed on the U95A chip, L3-unexposed explants showed an almost consistent basal expression of 3,243 genes and lack of expression of 5,560 genes across all time points (data not shown). Of these 12,000 genes, 157 genes were altered (41 induced, 116 repressed) after 72 h of explant exposure to L3. After 48 h of exposure to live L3, 57 genes were induced and 22 were repressed. Of the 128 genes with altered expression after 24 h of exposure to L3, 97 were induced and 31 were repressed in explants; only 30 genes were shown to change their expression pattern after 6 h of exposure to L3 (data not shown). L3-exposed explants down-regulated the expression of genes involved primarily in Ag processing and presentation. These genes, listed in Fig. 4, are as follows: proteosome subunit type (PSMA2); proteosome subunit 26S (PSMAD13); calpain, small subunit (CAPNS1); CLN2; and ubiquitination factor E4A (UBE4A). There was also down-regulation in the expression of lysosome-associated membrane protein 3 (LAMP3), which is DC specific, as well as expression of HLA-DMA. This finding correlates with the down-regulation of MHC class I and II on the cell surface of LC. Both the microarray data and the flow cytometry results suggest that the L3 stage of Bm may interfere with Ag processing and presentation in LC migrating from the skin (Figs. 3 and 4).
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Among the genes involved in the inflammatory response, IL-8 was the one chemokine that was consistently and markedly down-regulated by live L3 (Fig. 4). Down-regulation of IL-8 was of particular interest, because expression of this gene is highly up-regulated by L3 in macrophages (13) and by MF in both DC (12) and macrophages (our unpublished data). Down-regulation of IL-8 and HLA-DB1 by live L3 was also confirmed using real-time quantitative RT-PCR (Fig. 5). As seen, mRNA expression of both these genes in L3-exposed explants was inhibited by 5- to 7-fold when compared with L3-unexposed explants. Of interest, not all mRNA expression was inhibited, because IL-18 expression (for example) was induced by L3 1.8-fold.
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Because LC are capable of producing a large number of mediators including chemokines and cytokines, we assessed chemokine and cytokine expression broadly in L3-exposed and -unexposed epithelial explants. In supernatants collected at 72 h, there was no detection of any IL-12p40, IL-12p70, or IL-10 by unexposed or L3-exposed explants (data not shown). Furthermore, production of chemokines/cytokines known to be involved in maturation or migration of LC such as IL-16, IP-10, TNF-, GM-CSF, growth-related oncogene-, macrophage-inflammatory protein-3, TGF- (data not shown), or IL-8 (Fig. 6) was not significantly altered by live L3. Although 5 of 15 donors had a slight increase in IL-8 production after L3 exposure, for the group as a whole there was an insignificant change (geometric mean, 5.8% increase; p = 0.7) when compared with the L3-unexposed explant cells. Similar results were observed when L3 was physically separated from the explants using Transwells (data not shown). In contrast, the infective stage of S. mansoni induced IL-8 production in most donors (geometric mean, 38.5% increase) compared with unexposed cells. Notably, production of IL-18, a cytokine that induces LC migration through a TNF-- and IL-1-dependent mechanism (17), was up-regulated by live L3 (p = 0.016) and not by schistosomes (10 per explant; p = 0.1) (Fig. 6). Of interest, the L3-exposed but physically separated cultures had diminished IL-18 production compared with that of unexposed or L3-exposed (no Transwell) explants (data not shown).
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To assess the functional capacity of live L3-exposed LC, we examined whether the diminished expression of cell surface MHC class I and II translated into altered capacity of L3-exposed cells to activate CD4+ T cells in an allogeneic MLR compared with that of L3-unexposed cells (Fig. 7). As seen, L3-exposed cells had a diminished capacity (between 12 and 56% inhibition; p < 0.02) to induce CD4+ T cell proliferation in allogeneic MLR when compared with that of L3-unexposed cells. Furthermore, when L3 were separated from the explants by Transwells (Fig. 7, inset), the L3-exposed but physically separated crawl-out cells remained deficient in the induction of an allogeneic MLR. This suggests that secreted soluble parasite molecules (rather than direct L3/explant contact) are responsible for the functional impairment of the APC. Moreover, when a nonallogeneic system was evaluated in which autologous CD4+ T cells were cultured with L3-exposed or -unexposed LC in the presence of anti-CD3 at a 1:50 LC:T cell ratio, L3-exposed LC very clearly had a markedly diminished ability to activate CD4+ T cell proliferation (12–70% inhibition; p < 0.05). Autologous CD4+ T cells did not proliferate in response to either L3 Ag (likely due to the extremely low L3-specific T cell precursor frequency) or to another soluble Ag, tetanus toxoid. Moreover, in the presence of anti-CD3, CD4+ T cells activated with autologous L3-exposed LC had a diminished capacity to produce IFN- and IL-10 (Fig. 8). Of interest, IL-5 production in T cells induced by L3-exposed LC was variably affected (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LC have been shown to play a pivotal role in the induction of the immune response during the early phase of both intracellular (protozoan) and extracellular (helminth) parasitic infections. In leishmanial infections, for example, LC have been demonstrated to transport parasite Ag from the site of infection (skin) to the draining lymph nodes for primary activation of Leishmania-specific T cells (20). Such skin-penetrating pathogens can activate LC migration to the lymph nodes and may use intracellular replication within LC to evade the host immune response (21, 22). Furthermore, it has been suggested that resident LC may participate in the initiation of primary immune response in S. mansoni-infected guinea pigs, although the major APC involved were not the resident epidermal LC but rather blood-derived LC (9, 21). Moreover, in a model of schistosome infection, it has been demonstrated that LC are retained in the epidermis by a mechanism in which parasite-derived PGD2 impedes migration of LC (8).
To examine whether the L3 stage of the filarial parasite alters migration of LC from the epidermis, we exposed the explants to five live L3 for 72 h. Furthermore, in two experiments in which higher numbers of L3 (10 vs 5) were used, there was no difference seen in migration or cell surface molecule expression of crawl-out cells. We chose 72 h, because this is the time point when the maximum number of LC appears in culture. At this time point, cells harvested in culture are primarily LC, with between 20 and 39% contaminating KC (data not shown), and all of the L3 remain alive. These cells have the morphology of LC, with high surface expression of CD1a, MHC class I and II, and CD40, no expression of CCR5, and low expression of CCR6; however, these LC have a mature phenotype, expressing CCR7 and CD83. The heterogeneity seen in the ability of L3 to inhibit MHC class I and class II expression (Fig. 3) and in APC function (Fig. 7) could result from different numbers of LC vs KC in the crawl-out cultures; however, because of the limited number of LC for our experiments, it was not possible to perform simultaneously both flow cytometry and proliferation studies in the same donor.
These LC were clearly capable of taking up soluble L3 Ag or E/S products released from live L3 (Fig. 1). In addition, L3-exposed LC migrated from the epidermis in greater numbers than did LC that were not parasite exposed (1,800–25,000 cells/blister compared with 1,200–12,000) (Fig. 2), numbers that diminished if the L3 were separated from the explants by Transwells. This induced LC migration is likely related to the induction of mediators (e.g., cytokines/chemokines) involved in LC migration by KC or LC. Although proinflammatory mediators such as TNF- and IL-1 trigger mobilization of LC to lymph nodes, IL-18—another cytokine structurally similar to IL-1 (23, 24) and expressed by both DC and KC (25, 26, 27, 28)—has also been shown to mediate LC migration through mechanisms dependent on TNF- and IL-1 (17). Our finding in the present study that IL-18 (and not TNF-, IL-1, IL-16, TGF-, or IP-10) is preferentially induced by live L3 lends support to the concept that this cytokine may play a major role in the induction of LC migration in response to extracellular parasites. This migration of LC from the explants was not an effect of ongoing changes in the maturation state of these cells, because L3 did not appear to interfere with LC maturation. Intracellular protozoan pathogens have clearly been shown to induce increased expression of MHC class I and II molecules as well as other costimulatory molecules in mouse fetal skin DC (7). In some virally infected LC (HIV-1 (13), human papillomavirus (29)), however, surface expression of these molecules has remained unchanged. It is then even more interesting that live L3 caused diminished cell surface expression of MHC class I and II in most donors tested (Fig. 3) without altering expression of costimulatory molecules such as CD40, CD80, and CD86, suggesting that L3 fail to activate human LC. Our data find support in studies in which a cystatin homolog secreted by Bm also inhibits class II MHC-restricted Ag processing in a human B cell line (30). It has been shown that intradermal administration of IL-18 to mice resulted in a significant reduction in epidermal MHC class II LC densities (17). Whether the down-regulation of MHC class II in our system is due to the increased IL-18 remains to be tested. Notably, expression of a significant number of other genes involving Ag processing and presentation was also diminished by exposure to live L3 (Fig. 4) both by microarray analysis and by real-time RT-PCR (Figs. 4 and 5).
Pathology in lymphatic filariasis can be associated with acute inflammation (31), which in some situations has been related to production of proinflammatory cytokines in the lymphatic vessels (32). Moreover, Bm itself contains a homolog of human macrophage-inhibitory factor (33) known to activate human monocytes/macrophages to produce IL-8 and TNF- (34). In addition, filarial parasitic sheath proteins and live MF have been shown to induce IL-8 both in Hep2 cells (35) and in monocyte-derived DC. Thus, it is of interest that, in early infection, IL-8 is certainly repressed (see Figs. 6 and 7). This suggests that the parasite (at the site of infection) prevents the early accumulation of neutrophils (and other proinflammatory cells), a process that is overcome when infection becomes chronic and hosts become sensitized. Indeed, in a model of filaria-induced keratitis, both sensitization and IL-8 expression were required to produce the observed inflammation (36).
IL-12, a cytokine reported to be also produced by LC (37, 38), is known to provide a link between the innate and a Th1-adaptive immune response. In L. major infection of mice, IL-12p40 has been shown to be induced in the skin of these animals; furthermore, Leishmania-infected fetal skin-derived DC can release IL-12 (7); however, in the culture supernatant of unexposed or L3-exposed explants, neither IL-12p40, IL-12p70, nor IL-10 could be detected (data not shown). Brugia is not alone in this inability. It has also been shown that human papillomavirus-like particles fail to produce IL-12 (29). The consequence(s) of this lack of IL-12 production and the L3-induced down-regulation of LC MHC class I and II and genes involved in Ag presentation on T cell activation becomes the next step for investigation, because L3-exposed LC had a marked diminution in their ability to induce allogeneic or autologous CD4+ proliferation or cytokine production. In vitro down-regulation of cytokine production by CD4+ T cells is of importance, because it closely follows what has been found ex vivo in MF patients (39). That this inhibition can be achieved solely with E/S products (using Transwell to physically separate the worms from the explants) suggests that soluble parasite-derived molecules mediate the impairment of APC function. Furthermore, this inhibition appears to be stage specific in that when blisters were exposed to live MF or MF Ag, proliferation of autologous CD4+ T cells was not inhibited.
The targeting of LC for inhibition of an immune response is not necessarily a unique quality of the Bm L3 stage. Although precutaneous infection with S. mansoni leads to the activation of LC, these cells remain in the epidermis (8). Vaccinia virus abortively infects DC, blocks their maturation, and induces apoptosis to evade an immune response (40). Whereas human melanoma cells inhibit LC differentiation from CD34 precursors (41), human papillomavirus fails to activate human LC (29). Human CMV, adenovirus, and HIV each encode proteins that affect Ag processing and formation of MHC class I and II molecules (42, 43, 44). Therefore, it is possible that Bm has also evolved mechanisms to evade recognition by the immune system. The data presented in this study suggest that one such mechanism may be through functional alteration of LC, resident sentinels in the skin, such that they have a diminished ability to present Ag to T cells. This diminished function of the LC could allow the parasite to escape the barriers set up to contain the infectious stage, so that it can mature and develop and ultimately establish long-standing infections, with all of their attendant immunologic and medical consequences.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: L3, infective stage of Brugia malayi and human Langerhans cells; LC, Langerhans cell; KC, keratinocyte; MF, microfilaria; DC, dendritic cell; Bm, Brugia malayi.
Received for publication July 10, 2003. Accepted for publication March 8, 2004.
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References |
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 R. T. Semnani, P. G. Venugopal, C. A. Leifer, S. Mostbock, H. Sabzevari, and T. B. Nutman Inhibition of TLR3 and TLR4 function and expression in human dendritic cells by helminth parasites Blood, August 15, 2008; 112(4): 1290 - 1298. [Abstract] [Full Text] [PDF]
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S. Babu, C. P. Blauvelt, and T. B. Nutman Filarial Parasites Induce NK Cell Activation, Type 1 and Type 2 Cytokine Secretion, and Subsequent Apoptotic Cell Death J. Immunol., August 15, 2007; 179(4): 2445 - 2456. [Abstract] [Full Text] [PDF]
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 R. T. Semnani, P. B. Keiser, Y. I. Coulibaly, F. Keita, A. A. Diallo, D. Traore, D. A. Diallo, O. K. Doumbo, S. F. Traore, J. Kubofcik, et al. Filaria-Induced Monocyte Dysfunction and Its Reversal following Treatment. Infect. Immun., August 1, 2006; 74(8): 4409 - 4417. [Abstract] [Full Text] [PDF]
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J. D. Turner, R. S. Langley, K. L. Johnston, G. Egerton, S. Wanji, and M. J. Taylor Wolbachia Endosymbiotic Bacteria of Brugia malayi Mediate Macrophage Tolerance to TLR- and CD40-Specific Stimuli in a MyD88/TLR2-Dependent Manner J. Immunol., July 15, 2006; 177(2): 1240 - 1249. [Abstract] [Full Text] [PDF]
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M. D. Taylor, A. Harris, M. G. Nair, R. M. Maizels, and J. E. Allen F4/80+ Alternatively Activated Macrophages Control CD4+ T Cell Hyporesponsiveness at Sites Peripheral to Filarial Infection. J. Immunol., June 1, 2006; 176(11): 6918 - 6927. [Abstract] [Full Text] [PDF]
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K. H. Porthouse, S. R. Chirgwin, S. U. Coleman, H. W. Taylor, and T. R. Klei Inflammatory Responses to Migrating Brugia pahangi Third-Stage Larvae Infect. Immun., April 1, 2006; 74(4): 2366 - 2372. [Abstract] [Full Text] [PDF]
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S. Babu, C. P. Blauvelt, V. Kumaraswami, and T. B. Nutman Diminished Expression and Function of TLR in Lymphatic Filariasis: A Novel Mechanism of Immune Dysregulation J. Immunol., July 15, 2005; 175(2): 1170 - 1176. [Abstract] [Full Text] [PDF]
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 C. L. King and J. W. Kazura 3. UPSTREAM RESEARCH TO SUPPORT THE GPELF (BASIC RESEARCH): 3.1 PATHOGENESIS Am J Trop Med Hyg, November 1, 2004; 71(5_suppl): 29 - 33. [Full Text] [PDF]
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