Introduction
The data presented in chapter 4 showed that IL-6 could alter which determinants from HEL were selected for MHC class H-restricted presentation. This chapter aims to investigate what effect IL-6 has on dendritic cells that could account for this observation.
Chapter 1 discussed mechanisms of MHC class II restricited antigen processing and presentation. An antigen is captured, unfolded and usually partially degraded by proteolytic enzymes during endocytosis(Watts, 1997). MHC class II molecules are synthesised, assembled and transported through and out of the endoplasmic reticulum in association with invariant chain(Cresswell, 1994; Cresswell, 1996). In a specialised compartment or compartments maturing MHC class II on a secretory pathway meets antigen on an endocytic route(Cresswell, 1985; Neefjes e ta l, 1990; Peters e ta l, 1991). Here HLA-DM and HLA-DO or their murine counterparts regulate the loading of invariant chain peptide (CLIP)-bound MHC class II with peptide antigen(Denzin et al,
1997; Roche, 1995). The MHC class Il-peptide complex is eventually transported to and displayed at the cell surface. Additionally, some MHC class Il-peptide complexes can be formed independently of invariant chain, by loading onto recycling MHC class II in early endosomes(Pinet et al, 1995). It is important to note that most of the
experiments which have investigated the pathway of antigen processing have been performed with EBV immortalised B cell lines rather than non-clonal dendritic cells.
Antigen capture by [immature] dendritic cells occurs very differently compared to by B cells(Lanzavecchia, 1996). B cells capture antigen efficiently by their specific surface immunoglobulin, but very poorly by non-specific pinocytosis means(Lanzavecchia,
1985; Rock et al, 1984). In contrast, dendritic cells are highly pinocytic, in fact human dendritic cells are constitutively macropinocytic(Sallusto et al, 1995).
Macropinocytosis is a clathrin-independent process which internalises large volumes in vesicles up to 2|xm in diameter (Swanson and Watts, 1995). Once inside the cell, these
vesicles shrink and so concentrate the captured material(Racoosin and Swanson, 1993). Dendritic cells can also capture antigen by receptor-mediated endocytosis, using surface mannose or fucose receptors(Sallusto et al, 1995), FcyRII(Sallusto and Lanzavecchia,
1994) or DEC-205 receptors,(Jiang et al, 1995) and possibly with other as yet
uncharacterised receptors(Janeway, 1992). Receptor-bound antigen is then internalised via clathrin-coated pits, which are 20x smaller in diameter than
macropinosomes(Denzin et al, 1997; Roche, 1995). In the case of the mannose receptor, at endocytic pH the captured antigen dissociates from the receptor which recycles to the surface; other receptors are degraded along with their ligand(Sallusto et al, 1995). Dendritic cells also can capture larger objects such as latex beads or bacteria by phagocytosis, with or without opsonisation(Watts, 1997).
Macropinocytosis and mannose-receptor mediated uptake by dendritic cells occur by different mechanisms and the initial compartments into which the internalised materials enter by these two routes are different. However very soon after this initial event, endocytosed material via these two pathways meet in the same compartment(Tan et al,
1997). This compartment seems to be acidified in immature dendritic cells, which may facilitate the initial unfolding of protein antigen: intramolecular disulphide bridges are broken and the antigen is opened up for proteolytic attack(Lutz et al, 1997).
Subsequently, the antigen is shunted into a compartment containing (from Ira
Mellman's group) MHC class II, H2-M and lysosome associated membrane protein-2 (lamp-2)(Pierre et al, 1997) and/or (from Paola Ricciardi-Castagnoli's group) lamp-1 and cathepsin D(Lutz et al, 1997). This compartment is of higher pH than earlier endosomes(Lutz et al, 1997). Antigen can be retained for up to 2-3 days in this
compartment until the dendritic cell has matured(Pierre et al, 1997). This compartment may be an MHC class II loading site: MHC class Il-peptide complexes could be formed but not efficiently transported to the surface cell membrane until dendritic cell
maturation occurs(Pierre et al, 1997). Alternatively, processing of antigen and loading may be delayed until maturation, because the compartment is not acidified enough; low pH is required for both degradation of antigen and peptide loading (Lutz et al, 1997).
Maturation signals may then enable acidification, which would allow processing, loading and then presentation of MHC class Il-peptide complexes on the surface. In immature human dendritic cells MHC class Il-peptide complexes are transported to the cell surface, but are rapidly degraded. Maturation of these cells increases the half-life of antigenic complexes at the surface, from 10 hours to up to 100 hours (Celia et al, 1997).
The enzymes responsible for partially degrading antigen and generating T cell epitopes are not completely characterised, and are probably different for different antigens, in different cell types, and in different cell organelles. Candidate enzymes are cathepsin D(Rodriguez and Diment, 1992), cathepsin E(Bennett et al, 1992) and possibly cathepsin B(Van Noort et al, 1991).
Clearly, alterations in any aspect of this multi-component pathway, from rate of uptake to antigen unfolding and proteolysis to peptide loading and MHC class II transport could result in different determinants being selected from an antigen for
presentation(Sercarz et al, 1993). An investigation of this extremely complex network using nonclonal primary dendritic cells is difficult, and only a few parameters at a time can be measured, not all simultaneously.
The work in this chapter shows that interleukin-6 altered some but not all of these parameters in dendritic cells. The rate of uptake, exocytosis and the levels of processing enzymes were not changed after IL-6 treatment, but the early endosomal vesicles that antigen passed through were more acidic and contained lamp-1 in IL-6 treated dendritic cells. Degradation of protein antigen by control or IL-6 treated dendritic cells over time was not grossly different, but did vary slightly. Finally, differential display of
messenger mRNA from IL-6 and non-IL-6 treated dendritic cells revealed distinct patterns of gene expression in the two cell types. How these findings may explain the observed differences in HEL processing is discussed, and ways in which this issue might be further investigated are outlined.
Results
Endocytic progress of captured dextran through a dendritic cell is not altered by IL-6
The first event of antigen processing is antigen capture and endocytosis. As discussed earlier, IL-6 did change the rate of uptake of either fluid-phase or mannose receptor (MR) mediated uptake by dendritic cells. However this does not mean that the route antigen takes once inside the cells would be the same. This was investigated by
following the location of Texas Red-conjugated dextran (TR-Dx) within the cells during endocytosis. IL-6 treated cells and controls were incubated with 1 mg/ml of TR-Dx on ice for 5 minutes to allow binding by MR without internalisation, then washed and resuspended in medium at 37°C so that endocytosis could then progress. Aliquots of cells were removed after various times, washed, fixed and mounted on poly-L-lysine coated slides and viewed on a confocal microscope. Representative cells are shown in figure 5.1.
As mentioned above, although dextran and HEL would be captured by dendritic cells using MR and pinocytosis respectively, they reach identical compartments 5 minutes after internalisation. So although the pictures shown here strictly relate only to MR mediated uptake of dextran, it is very likely that matching events would occur with endocytosed HEL. Doing similar pulse-chase experiments with fluorochrome labelled HEL is difficult because the rate of initial capture by pinocytosis is not as fast as the on- rate of the MR, so the pulse for HEL would need to be for longer (30 minutes), and could not be at 4°C as for the MR. The results from this type of experiment are unclear (P. Medd, personal communication) because at the end of the pulse, dendritic cells would have pinocytosed some HEL for 30 minutes, but some would have only just been internalised. So within any given cell, internalised HEL molecules would be at different stages on the endocytic route. In contrast, using cold-pulsed TR-Dx followed by a warm chase, all the TR-Dx starts off on the surface and is then internalised together.
Figure 5.1
Location of Texas-Red labelled dextran by endocytosed by dendritic cells for 5 minutes (a, b) or 60 minutes (c, d). Cells in a, c and d are 9pm in diameter, bar in b=10pm.
Figure 5.1 shows that dextran was initially internalised all around the circumference of the cell after 5 minutes, but after 1 hour almost all the dextran was located in a large intracellular compartment or compartments, which was generally perinuclear. At intermediate timepoints, dextran was located between these two areas in vesicles that became progressively less discete as time went on (not shown). It appeared that these vesicles met to form the large compartment, reminiscent of that observed in an
significant differences between IL-6 treated and control dendritic cells in the route by which dextran was endocytosed, for example the large compartments were of a similar size, and the rate of progress of dextran to this compartment from earlier ones was the same. This does not mean however that the compartments that dextran passes through on the endocytic route have the same characteristics in both cell types.
Characteristics offluorochromes at different pH
Endocytosis by dendritic cells was further investigated using fluorochrome-labelled markers (table 5.1). The fluorescence of some of these markers is influenced by vesicular pH. This enabled study of vesicle acidification, which is essential for antigen processing.
Marker type Route of uptake pH sensitivity
Lucifer Yellow Fluid endocytosis Not sensitive
HEL-FITC Fluid endocytosis Sensitive
FITC-dextran Mannose receptor / fluid endocytosis
Sensitive
TRITC-dextran Mannose receptor / fluid endocytosis
Not sensitive
FITC+TRITC-dextran (double label)
Mannose receptor / fluid endocytosis
FITC is sensitive, TRITC is not
Table 5.1
Characterisitics of markers used for endocytosis assays. FITC=fluorescein isothiocyanate, TRITC=tetramethylrhodamine isothiocyanate.
The fluorescence of FITC alters depending on the pH of its environment. Protonation of dianionic FITC to monoanionic FITC, which occurs at about pH 6.5 when FITC is conjugated to dextran, quenches emission at the wavelength maximum (see figure 5.2). When a FACS can or similar instrument is used to analyse cells which have taken up a
FITC-conjugated marker, the fluorescence detector collects radiation in the region of 515-545 nm (the emission maximum of dianionic FITC excited at 488nm is 519nm). So if FITC-dextran is in an endosome of sufficient acidity to protonate dianionic FITC, the cells will fluoresce less. This property of FITC, which does not occur with TRITC or Lucifer Yellow at physiological pH, can be used to calculate the acidity within vesicles. In the experiments shown here these markers were used to compare the relative acidity of vesicles during endocytosis by IL-6 treated or control dendritic cells.
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Q) CC E UJ IL. i i 8 9 2 3 4 5 6 7 pH Figure 5.2A: Protonation of FITC^" to FITC". B: Change in fluorescence emission maximum of FITC-dextran (FEm) intensity with pH . Figure adapted from (Ohkuma, 1989). At pH about 4.4 FITC" is protonated again to become a neutral molecule.
IL-6 treated dendritic cells quench the fluorescence o f endocytosed pH sensitive markers
The fluorescence of dendritic cells endocytosing Lucifer Yellow (LY) or HEL-FITC (made using a FITC conjugation kit from Sigma) was measured after various times of exposure to the endocytic marker. Figure 5.3 shows that IL-6 treated dendritic cells and control dendritic cells taking up LY fluoresced equally over time, but that IL-6 treated dendritic cells appeared to take up less HEL-FITC than control dendritic cells. This is unlikely to be the explanation, as both LY and HEL-FITC are taken up in a similar way.
200
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100 O) in GM-CSF GM-CSF + IL-6 0 60 120 180 150 1 100 ■ 60 120 180 Time (m inutes) Figure 5.3IL-6 treated dendritic cells taking up HEL-FITC fluoresce less than control dendritic cells, while fluorescence of LY is unaffected. 10^ cells were incubated with LY or HEL-FITC at 1 mg/ml in 96 well plates at 37°C for the times indicated, then put on ice, washed in cold PBS twice, 10^ cells were analysed by FACS, and the median
fluorescence was calculated. The median fluorescence of cells incubated with 1 mg/ml of marker at 4°C for 30 minutes was subtracted from other values.
HEL is a polar molecule, so the attraction of HEL-FITC to cells is influenced by the electrostatic charge of cell membranes. It was possible that IL-6 treatment of dendritic cells might alter this attraction, so that less HEL was taken up by these cells while
uptake of LY was unaffected. Alternatively, IL-6 might act to acidify endocytic vesicles which quench FITC fluorescence. Two experiments favour the latter option. If IL-6 treated dendritic cells endocytosing HEL-FITC were fixed after each time point, the fluorescence of the cells increased, whereas the fluorescence of control cells taking up HEL-FITC or of either cell taking up LY did not increase as much. Fixation equilibrates intracellular pH gradients with the pH of the assay medium. See figure 5.4.
LY HEL-FITC 300 200 E c O) LO 100
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o S 200 100 0 1 2 3 4 300 200 100 O - GM -CSF # - GM -CSF fixed 1 2 3 4 0 Marker concentration (mg/ml) Figure 5.4Fixation of IL-6 treated dendritic cells after uptake of HEL-FITC increases their fluorescence. 10^ cells were incubated with various marker concentrations for 30 minutes at 37°C, put on ice, washed twice, resuspended in PBS with or without 1% paraformaldehyde, and analysed as for figure 5.3. Fixed cells with zero marker did not fluoresce more than unfixed cells.
The second experiment favouring different pH as an explanation for lower FITC fluorescence in IL-6 treated cells used fluorochrome-labelled dextrans as markers of endocytosis (figure 5.5). Dextrans are primarily taken up via the mannose receptor by mouse dendritic cells, in fact preblocking the mannose receptor with mannosylated BSA reduced the uptake of dextrans by 70% (not shown). When dendritic cells were incubated with dextran doubly labelled with FITC and TRITC, IL-6 treated dendritic cells fluoresced less at the FITC emission wavelength maximum than control cells at early time points, but both cell types fluoresced equally at the TRITC emission
maximum wavelength. As uptake of dextran by the mannose receptor is not affected by the charge of the membrane, and as a similar quenching effect is seen as with HEL- FITC, a change in pH after IL-6 treatment would appear to be responsible for the pH quenching of both FITC-conjugated markers.
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120 90 O 3 60 _l LL C <2 % o S GM-CSF GM-CSF+IL-6 30 0 30 60 90 120 120 90 60 GM-CSF GM -CSF+IL-6 30 0 30 60 90 120 0s
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Q) Time (minutes) Figure 5.5IL-6 treated dendritic cells quench fluorescence of FITC (A), but not of TRITC (B), during endocytosis of double-labelled dextran conjugated to both fluorochromes. Experiment performed as for figure 5.3. Similar results were obtained using singly labelled dextrans.
The biggest difference in FITC fluorescence was at early time points, when all of the internalised material would be expected to be in early endocytic compartments. Later, some material would also be in these compartments (as these are not pulse-chase experiments), but some would be in later vesicles, which may not be as acidic. So the FITC fluorescence at a given time is determined by both the amount of marker taken up and the distribution of markers in vesicles of varying acidity. To analyse further the role of vesicles in determining FITC fluorescence, a pulse-chase experiment was carried out. Cells were incubated in the cold with doubly labelled dextran, washed, then incubated at 37°C for various times. At each time point, cells were removed to ice, either fixed or not, and then analysed by FACS for FITC and TRITC fluorescence. See figure 5.6.
280 GM-CSF GM-CSF FIX -J U. c <0 ’"B 0) S 2 3 0 - 180 120 240 360 0 280 GM -CSF+IL-6 GM -CSF+IL-6 FIX 230 180 0 120 240 360 220 1 LL 1 7 0 - 120 0 120 240 360 220 170 120 120 240 360 0 Time (min) Figure 5.6
FITC (A, B) or TRITC (C, D) fluorescence of control (A, C) or IL-6 treated (B, D) dendritic cells coated on ice with doubly labelled dextran and then allowed to endocytose (and exocytose) dextran at 3TC over time. Analysis as for figure 5.5.
These results shows that as captured dextran is endocytosed through the cell, the FITC fluorescence is quenched at early time points in both control and IL-6 treated unfixed cells (5.6a, b), although the effect is much bigger with IL-6 treated cells. Fixation of cells dequenches the FITC fluorescence. At the same time points, TRITC fluorescence is unaffected by IL-6 treatment or fixation. At later time points, FITC fluorescence does not seem to be quenched by IL-6 treated cells, but is by control cells as fixation
increases their fluorescence, suggesting that the later compartment is more acidic in control cells than for IL-6 treated cells. As time progresses, FITC and TRITC
fluorescence decreases for both cell types, showing exocytosis of the dextran. However the fluorescence at the final time point is still more than for cells with no marker, indicating that a certain level of dextran is retained. The data in this experiment are consistent with the idea that IL-6 treated dendritic cells, while endocytosing very similarly to control cells in terms of the rate of uptake, route their captured material through more acidic vesicles than control cells in the first 30 minutes after
internalisation.
Visualisation o f acidic vesicles in dendritic cells and colocalisation with endocytosed material
The location of acidic endocytic vesicles can be identified in cells using DAMP, which is a conjugate of dinitrophenol (DNP) linked to a weak base (figure 5.7)(Anderson et al, 1984). This molecule has been used to study vesicle acidification in cystic
fibrosis(Barasch et al, 1991), and in an immature dendritic cell line(Lutz et al, 1997). NO,,
CHg
NH(CH2)^(CH2)gNH2
.2 HCI
Figure 5.7
Structure of DAMP (N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-
DAM P can pass through membranes, permeates through the cell and accumulates (after
protonation) in acidic vesicles. C ells are then fixed, permeabilised, incubated with anti-
DNP antibodies, which are visualised by fluorochrome labelled second layer antibodies
and analysis on a confocal m icroscope. This method was used to locate acidic vesicles
in IL-6 treated and control dendritic cells, shown in Figure 5.8. This figure show s that