Uptake and intracellular trafficking of superantigens in dendritic cells

Abstract

Bacterial superantigens (SAgs) are exotoxins produced mainly by Staphylococcus aureus and Streptococcus pyogenes that can cause toxic shock syndrome (TSS). According to current paradigm, SAgs interact directly and simultaneously with T cell receptor (TCR) on the T cell and MHC class II (MHC-II) on the antigen-presenting cell (APC), thereby circumventing intracellular processing to trigger T cell activation. Dendritic cells (DCs) are professional APCs that coat nearly all body surfaces and are the most probable candidate to interact with SAgs. We demonstrate that SAgs are taken up by mouse DCs without triggering DC maturation. SAgs were found in intracellular acidic compartment of DCs as biologically active molecules. Moreover, SAgs co-localized with EEA1, RAB-7 and LAMP-2, at different times, and were then recycled to the cell membrane. DCs loaded with SAgs are capable of triggering in vitro lymphocyte proliferation and, injected into mice, stimulate T cells bearing the proper TCR in draining lymph nodes. Transportation and trafficking of SAgs in DCs might increase the local concentration of these exotoxins where they will produce the highest effect by promoting their encounter with both MHC-II and TCR in lymph nodes, and may explain how just a few SAg molecules can induce the severe pathology associated with TSS.

Figure 1. SAg incorporation in DCs and cell activation markers.

Bone marrow DCs were pulsed 1 h with SEG-FITC, washed, and immunolabeled with anti CD11c-PE. (A) Percentage of DCs incorporating different concentration of SEG-FITC at 37°C and 4°C vs. non-pulsed DCs as control. (B) Percentage of DCs showing inhibition of SEG-FITC internalization by wortmannin (WT) and 5-ethyl-N-isopropyl amiloride (EIPA) added at final concentrations of 100 nM and 50 µM, respectively, 15 min before SAg-FITC at 50 µg/ml. (C) Representative histograms of the phenotype of DCs in the presence of SAgs. (D) Cell surface MHC-II molecules on DCs treated with SSA, SEG, SEI or LPS for 24 h, compared to non-treated basal control, and expressed in MFI. (E) CD80 on SAgs- or LPS-treated DCs compared to non-treated. (F) Endocytosis of OVA-FITC by SAg or LPS pre-treated DCs. (G) DCs were incubated for 1 h with SEG at 37°C or 4°C as control, washed, and cultured for additional periods from 0 to 24 h. At each endpoint, cells were washed and disrupted in order to isolate the endosomal/lysosomal compartment. These fractions were incubated with mouse splenocytes to assess proliferative activity by ³H-Thy incorporation in triplicate. The proliferation index (PI) was calculated as cpm/basal cpm. Basal represent the proliferation of T cells stimulates by DCs incubated in the absence of SAg. (H) SEG-pulsed DCs cultured for additional periods of 0 to 24 h. At each endpoint, cells were washed and immunolabeled with mouse anti-SEG polyclonal antibodies and anti-mouse-FITC, as second antibody to evaluate the presence of SEG on the cell surface. Figures show a representative experiment of 3–5. *p<0.05, **p<0.01.

Figure 2. After incorporation in DCs, SEG co-localize with LysoTracker in acidic organelles.

DCs were cultured on poly-L-lysine treated slides, pulsed 1 h at 37°C with 50 µg/ml SEG-FITC and 50 µM Lysotracker RED (Molecular Probes, Invitrogen), washed and cultured further for different time periods (0–120 min). Fluorescently labeled cells were fixed with paraformaldehyde at 4°C, and cover slides mounted using anti-fade mounting media (Vector Laboratories). Image capture was performed using a Nikon C1 confocal laser scanning microscope with a PlanApo 60× Oil AN1.40 lens. For detection of FITC a 490 nm laser was used. A 546-nm excitation wavelength and a 590-nm emission wavelength were used for LysoTracker. Analysis by confocal microscope (6000×) shows that SEG barely co-localized at acidic compartment at time point 0, but clearly co-localized with LysoTracker at the 60 min time point and later. Cells pulsed with 50 µg/ml OVA-FITC for 1 h showed co-localization of OVA with Lysotracker at time point 0, but 60 min later only LysoTracker remained inside the cell. Figure shows a representative experiment of 3.

Figure 3. After incorporation in DCs, SEG is located in vesicles inside DCs and then is exposed on cell membrane.

DCs were cultured on poly-L-lysine treated slides, pulsed 1 h at 37°C with SEG, washed, and cultured further for different time periods (0–240). Indirect Immunolabeling of intracellular SEG and the organelle-specific markers was conducted with mouse polyclonal serum to SEG concurrently with polyclonal rabbit anti-EEA1, anti-RAB-7 or anti-LAMP-2, (Sigma Aldrich, St. Louis, MO). Cells were then incubated with FITC-labeled anti-rabbit and rodamine A-labeled anti-mouse for analysis by confocal microscopy. Image capture was performed using a Nikon C1 confocal laser scanning microscope with a PlanApo 60× Oil AN1.40 lens. For detection of rhodamine, a 543 nm laser and 490 nm laser for FITC were used. Controls of non-pulsed cells were performed in parallel. (A) Immediately (0 min) and 20 min after the 1 h pulse, the location of SEG (red) was exclusively intracellular and showed strong co-localization with EEA1 (green), which was lost at 40 min of culture. At this latter time, SEG was still inside vesicles, but EEA1 was scattered throughout the cell cytoplasm. (B) Co-localization of SEG with RAB-7 starts at 20 min after 1 h pulse with SAg. At 60 min, a portion of SEG could be seen in RAB-7⁺ small vesicles scattered throughout the cytoplasm and also concentrated in clusters near the cell membrane. One hundred and twenty min after pulse, SEG was seen in RAB-7 negative vesicles. (C) Only 20 min after the 1 h pulse, SEG could be detected in the lysosomal compartment surrounded by LAMP-2). At 60 min, co-localization with SEG was also apparent. However, at 120 min, there was only trace co-localization of LAMP-2 and SEG, but clusters of SAg could be seen on the cell membrane, which remained up to 240 min of culture. Figures show a representative experiment of 4–5.

Figure 4. SEG co-localizes with MHC-II at the cell membrane and in vesicles.

DCs were cultured in poly-L-lysine treated slides, pulsed 1 h at 37°C with SEG-FITC, washed four times and cultured for different periods (0–240 min). Cells were then fixed with paraformaldehyde and permeabilized with saponin for immunolabeling with a phycoerythrin-Cy5-labeled monoclonal antibody against mouse I-A and I-E MHC-II. A control of non-pulsed cells was performed in parallel. Confocal images were captured using a PlanApo 60× Oil AN1.40 lens. After 1 h pulse, SEG showed a dispersed pattern, with some clusters, near the cell membrane. Strong staining of MHC-II was observed at a similar location. At 60 min, SEG co-localized with MHC-II in vesicles. One hundred and eighty min later, clusters of MHC-II and SEG were observed, in addition to a scattered pattern for both SEG and MHC-II close to the cell membrane. Figure shows a representative experiment of 5.

Figure 5. Analysis of SAg uptake by DCs and release into the medium by SPR.

DCs were incubated in the presence, or absence, of 100 ug/mL SEG for 1 h, washed four times, and cultured in fresh medium for 3 h (SNSEG). Supernatant SNSEG diluted two-fold in fresh medium, or DC culture medium (CM) were injected over immobilized TCR Vβ8.2 and responses recorded as a function of time for 60 sec (30–90 sec), and then buffer was injected for dissociation. In addition, SNSEG diluted two-fold in fresh medium were neutralized with mouse anti-SEG (SNSEG+Ab) and injected over immobilized TCR Vβ8.2. Inset: Immunoblot of supernatant from DCs incubated in the presence (SNSEG) or absence (CM) of SEG. MWM: molecular weight markers. Figure shows a representative experiment of 3.

Figure 6. DCs induce superantigenic response after SEG uptake.

DCs were pulsed 1 h at 37°C or 4°C with SEG, washed, and co-cultured with isogenic splenocytes in triplicate; controls of DCs with no addition of splenocytes were conducted in parallel. (A) ³H-Thy incorporation for DCs, splenocytes, and DCs plus splenocytes. (B) DCs were pulsed with different concentrations of SEG for a dose response curve, and co-cultured with splenocytes in triplicates. (C) Splenocyte proliferation induced by SEG-pulsed DCs and SEG-pulsed DCs in the presence of Wortmannin, performed in triplicates. (D) Rabbit anti-SEG serum was added to the cultures of pulsed DCs and splenocytes to specifically block SEG function. (E) Splenocytes were cultured in the presence of 0.01 µg/ml SEG or 1 µg/ml ConA, with the addition, or not, of different dilutions of rabbit anti-SEG serum. One representative experiment is shown of 3 to 5 experiments. **p<0.01.

Figure 7. After SEG uptake, DCs stimulate lymphocytes bearing Vβ8.2 TCR.

(A) DCs were pulsed 1 h at 37°C with SEG, washed, and co-cultured with isogenic splenocytes. FACS analysis of T cell populations was conducted with PE-conjugated anti-Vβ8.2+1 TCR and PE-conjugated anti-Vβ8.3 TCR monoclonal antibodies (BD Pharmingen). A control of non-pulsed DCs and splenocytes was conducted in parallel. (B) DCs pulsed at 37°C or 4°C with 50 µg/ml SEG were injected intradermically into BALB/c mice foot pads. Control animals were injected with non-pulsed DCs in parallel. FACs analysis with anti-Vβ8.2+1 and anti-Vβ8.3 was conducted on cells recovered from popliteal lymph nodes after 48 h. One representative experiment is shown of 3. *p<0.05 and ** p<0.01. (C) Schematic diagram depicting SAgs uptake and trafficking in DCs. After incorporation by macropinocytosis, SAgs can be located in early and late endosomes and lysosomes, as biologically active molecules, and then are recycled to the cell membrane. After trafficking, SAgs are capable of stimulate T cells bearing the proper TCR, triggering lymphocyte proliferation.