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IMMUNOBIOLOGY
From the Unit of Protein Biology, Laboratory of
Immunology, and Biotechnology Section of Roma, National Cancer
Research Institute, 16132 Genoa, Italy; Department of Experimental
Medicine and Pathology, University of Rome "La Sapienza," Rome,
Italy; Istituto Dermatologico San Gallicano IRCCS of Rome, Italy;
Laboratory of Tumor Immunology, San Raffaele Scientific Institute,
Milan, Italy.
We recently reported that human dendritic cells release the
leaderless secretory protein interleukin-1 Regulated secretion is traditionally
considered as a specialized process occurring in only a few polarized
cell types, namely, exocrine, endocrine, and neuronal
cells.1 However, former observations that, on binding to
antigen-presenting B cells, T-helper cells release lymphokines
preferentially over the membrane area where T-cell receptor
cross-linking is occurring,2,3 led to the hypothesis that
a regulated polarized secretion may exist also in nonpolarized cells.
It is now clear that many hemopoietic cells use regulated secretion;
examples are mast cells and granulocytes, which degranulate in response
to Fc receptor cross-linking,4 or platelets, which respond
to vascular lesions by releasing small molecules and proteins from
intracellular granules.5 Unlike conventional secretory cells, endowed with specific structures for storage and release, hemopoietic cells use secretory lysosomes, a mixture organelle between
lysosomes and secretory granules.6 Interestingly, other cell types, including fibroblasts, are able to transform conventional lysosomes into a secretory organelle underlying inducible
exocytosis7; thus, regulated secretion seems to be a more
widespread phenomenon than previously thought. However, even if
secretory lysosomes are quite ubiquitous, the ability of directing
their content toward a given target, resulting in polarized secretion,
has been described so far only for a few immune cells, namely, T
lymphocytes or natural killer cells.8,9 This may depend on
how exocytosis is induced. Indeed, in most cell types exocytosis is
driven by stimuli triggering structures distributed uniformly on the
plasma membrane (such as IgE receptors on mast cells); in contrast, in
the case of T and natural killer cells, binding to target cells with
engagement of a single or few receptor complexes results in local
activation leading to polarized degranulation.
Although it is known that increases in intracellular free calcium
concentration ([Ca++]i) and cytoskeleton
rearrangement occur during the process,7 the molecular
mechanisms underlying regulated lysosome exocytosis are still unclear.
We have recently demonstrated in monocytes an adenosine triphosphate
(ATP)-dependent exocytosis of secretory lysosomes, resulting in
release of lysosomal enzymes and of the mature form of interleukin-1 Derivation of DCs from adherent cells
Establishment of mixed lymphocyte reaction, enrichment of
CD4+ or CD8+ T cells
Cytolytic assay and cytoplasmic immunofluorescence Cytolytic activity of 7-day or, as a positive control, 18-day alloreactive T cells against DCs was tested in a 51Cr release assay as described.18 Briefly, DCs were loaded with 51Cr and cocultured for 4 hours with alloreactive T cells, used as effector cells at an effector/target ratio of 10:1. Results are expressed as percentage of cytotoxicity as described.18 T cells from 7-day MLRs were also tested for their content in intracellular perforin by cytoplasmic immunofluorescence using the antiperforin mAb G9 (Pharmingen, San
Diego, CA), before or after 6 hours of contact with DCs.
Culture conditions The DCs, prepared as above, were incubated alone or with allospecific T cells from 7- day MLRs (whole T cells or CD8+ or CD4+ purified T cells) at a T cell/DC ratio of 10:1.16 Culture of DCs alone or cocultures with T cells were carried out for different periods of time, up to 6 hours. When indicated, ionomycin (1 mM, Sigma) or the L-type calcium channel19 agonist BayK 8644 (10 µM, Sigma) was added for the last 10 minutes, or thapsigargin (10 nM, Sigma) for the last 30 minutes. In other experiments, the L-type calcium channel blocker nifedipine (10 µM, Sigma) or the calcium chelator ethylene glycol-bis( -aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA, 5 mM, Sigma) was present during the entire duration of coculture. At the
end of the incubations, supernatants were concentrated by 10%
trichloroacetic acid (TCA); cells were lysed in 1% Triton X-100
(Bio-Rad, Milan, Italy) containing buffer.16 In other
experiments, DCs were pretreated with nocodazole (Sigma) for 1 hour at
30 µM and cocultured for 6 hours with alloreactive T cells at a T
cell/DC ratio of 10:1, in the presence of 20 µM nocodazole. As a
control, DCs were fixed for 10 minutes with 1% glutaraldehyde (Sigma)
prior to 6 hours of coculture with T cells.
Subcellular fractionation by differential ultracentrifugation and Percoll density gradient Subcellular fractionation was carried out as described by Pitt and coworkers20 with slight modifications.10 Briefly, cells were washed, resuspended in homogenizing buffer (250 mM sucrose, 5 mM EGTA, 20 mM Hepes-KOH, pH 7.2) at 5 × 107/mL and broken in a Dounce homogenizer. Unbroken cells, debris, and nuclei were discharged by 3 cycles of centrifugation at 800, 1000, and 1200g, and the postnuclear supernatant (PNS) obtained was diluted 10-fold in homogenizing buffer and centrifuged at 35 000g for 1 minute. The pellet was kept as P1; the P1 supernatant was centrifuged at 50,000 g for 5 minutes, leading to a second pellet (P2). The 2 pellets were treated with 0.1 mg/mL proteinase K (Sigma) for 30 minutes on ice (to remove cytosolic pro-IL-1 possibly bound to the external membrane of the organelles)
in the presence or absence of 0.1% Triton X-100 followed by addition of protease inhibitors (Sigma). The P2 supernatant was spun at 100,000g for 30 minutes, and the resulting supernatant was
kept as cytosolic fraction and concentrated by 10% TCA precipitation. P1 and P2 pellets obtained as above were resuspended in 1 mL of a
buffer containing 3 mM imidazole and 250 mM sucrose at pH 7, and mixed
with 9 mL of the same buffer containing Percoll (Sigma) up to
25%.10,21,22 After centrifugation for 2 hours at
90 000g (Beckman TiSW41 rotor, 27 000 rpm), fractions were
collected using a needle connected to a peristaltic pump (Amersham
Pharmacia Biotech, Milan, Italy), membranes were lysed with 0.5%
Triton X-100, diluted 5-fold in the same buffer, ultracentrifuged 30 minutes at 100 000g to remove Percoll,10,21,22
and concentrated by TCA precipitation.
Western blot analysis Aliquots of cell lysates corresponding to 0.5 × 105 DCs (or 100 µg proteins; protein dosage performed with the Bio-Rad kit based on the colorimetric Lowry method) and the correspondent TCA-concentrated supernatants, or aliquots of PNS, TCA-concentrated cytosol (100 µg proteins) and the corresponding whole P1, P2, or aliquots from the different Percoll gradient fractions were solubilized in reducing sample buffer and resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions.10 Gels were electrotransferred onto nitrocellulose filters (Hybond ECL, Amersham Pharmacia Biotech), stained with Ponceau S (Sigma) to confirm equal protein loading (not shown), and destained prior to blocking overnight with 10% nonfat dry milk in phosphate-buffered saline (PBS). Filters were hybridized with the antihuman IL-1 mAb 3ZD (IgG1, provided by National Cancer Institute Biological Resources Branch, Frederick, MD) or the antihuman cathepsin D mAb (IgG2a, Calbiochem, Milan, Italy), followed by a
horseradish peroxidase (HRP)-conjugated goat antimouse IgG (Dako, Milan, Italy), or with the rabbit antihuman Rab7 antibody (gift of S. Méresse, Marseille, France23), followed by an
HRP-conjugated goat antirabbit IgG (Dako) and developed by ECL-Plus
(Amersham Pharmacia Biotech) according to the manufacturer's
instructions. When stated, densitometric analyses of the blots were
performed.10
Conventional and immunoelectron microscopy The P1 and P2 fractions were processed for postembedding immunocytochemistry as described.10,24 Briefly, fractions were fixed in 1.0% glutaraldehyde (Gibco Laboratories, Grand Island, NY) in PBS for 1 hour at room temperature, partially dehydrated in ethanol, and embedded in LR White resin (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were collected on nickel grids and processed for double immunolabeling; sections were incubated first with anti-IL-1 mAb (IgG1) followed by 10 nM goat antimouse IgG
gold-conjugated (British Biocell International, Carditt, United
Kingdom), postfixed with 1.0% glutaraldehyde for 10 minutes to prevent
interference among different antibodies and gold
conjugates,25 and then incubated with rabbit anticathepsin D antibody (Upstate Biotechnology, Lake Placid, NY) followed by 18 nm
protein-A gold prepared by the citrate method.26 Control experiments were performed using as primary antibodies an unrelated isotype-matched (IgG1) mAb and a rabbit preimmune serum (both kindly
provided by Dr A. Santoni, Rome, Italy). All sections were finally
stained with uranyl acetate and lead citrate. DCs interacting with
CD4+ or CD8+ T cells for various periods of
time were fixed in glutaraldehyde as above and were processed for
conventional thin section electron microscopy as
described.24 Thin sections were examined unstained and
poststained with uranyl acetate and lead hydroxide. Quantitative evaluation of immunolabeling was performed by comparing the number of
small (10 nm) and large (18 nm) gold particles present inside organelles displaying the ultrastructural appearance of late
endosomes/lysosomes. Fifty images of each type of structure, randomly
photographed from 3 different immunolabeling experiments, were analyzed.
Single cell analysis of calcium fluxes by video-microscopy and ratio-imaging Single cell analysis of calcium fluxes was performed as described.27 CD4+ or CD8+ alloreactive or nonspecific T cells were loaded 1 hour at 37°C with 1 µM FURA 2-AM (Sigma) and added to DCs, similarly loaded with FURA 2-AM, cultured on round coverslips, placed in a microincubator (Medical System, Greenvale, NY) on an inverted epifluorescence Axiovert 10 microscope (Zeiss, Oberkochen, Germany) and maintained at 37°C by a temperature controller (TC-202, Medical System). FURA 2-AM was excited with a high-pressure 75 W xenon arc lamp fitted with appropriate filters on a shutter controlled by a Pentium 90 MHz computer. Excitation light was at 334 and 380 nm; emitted light was filtered at 510 nm. Two 334:380 ratios were taken each second and video images collected with an intensified charged coupled device camera (Atto Instruments, Rockville, MD) and recorded every 15 seconds using the image-processor program Attofluor RatioVision 6.08 (Atto Instruments). Results were stored as ratio of FURA 2-AM fluorescence at 334 nm divided by the fluorescence at 380 nm excitation. The [Ca++]i was calculated according to Grynkiewicz and colleagues.28 [Ca++]i increases were measured on interaction of DCs with CD4+ or CD8+ and nonspecific or specific T lymphocytes. The integrity of DCs after coincubation with T cells was controlled by evaluating the maintenance of FURA 2-AM fluorescence.
IL-1 synthesis but not secretion. In contrast,
CD8+ allospecific T lymphocytes do not induce per se
pro-IL-1 synthesis, but drive processing and secretion of the
bioactive cytokine by mature DCs.16
Noteworthy, IL-1
Pro-IL-1 and cathepsin D prompted us
to investigate whether the 2 proteins colocalize in organelles belonging to the endolysosomal compartment. LPS-activated DCs were
cultured for 4 hours with or without alloreactive
CD8+ T cells, and the PNSs obtained at the end
of the culture period were subjected to 2 sequential
ultracentrifugations, giving rise to 2 pellets, P1 and P2, enriched in
lysosomes and endosomes, respectively.10,20 As shown in
Figure 2A, DC pro-IL-1 is detected in
both P1 and P2 (lanes 3 and 4). Comparison with the pro-IL-1 band
present in the cytosolic fraction (lane 1) allowed the calculation that
particulated pro-IL-1 ranged, in the different experiments, from
10% to 20% of the total cellular pro-IL-1. Interestingly, the P1
fraction from DCs cultured with alloreactive T cells (Figure 2B, lane
3), contained also a 29-kd IL-1 band of intensity similar to the
35-kd pro-IL-1 band. This band, which is almost undetectable in the
cytosolic fraction (lane 1), corresponds to the first product of ICE
processing, is absent in cells treated with ICE
inhibitors,16,30 and is also secreted in large
amounts by DCs cultured with alloreactive T cells (Figure 1A). These
observations suggest that pro-IL-1 present in P1 is the direct
precursor of secreted IL-1.
To clarify whether IL-1
Cathepsin D and IL-1 secretion is triggered by extracellular
ATP.10,31-33 In contrast, although DCs express purinergic
receptors,14 ATP increased neither IL-1 (Figure
4A) nor cathepsin D (Figure 4B) secretion
by mature DCs cultured alone (Figure 4, lanes 1 and 2) or with
alloreactive T cells (compare lanes 3 and 4). Because exocytosis of
secretory lysosomes is a Ca++-regulated
process,7,34,35 and secretion by DCs of a number of
cytokines, including the leaderless secretory protein IL-18, is
Ca++ dependent,27,36 we investigated whether
IL-1 and cathepsin D secretion is induced in DCs by increases in
[Ca++]i. As shown in Figure
5A, 10-minute ionomycin stimulation of mature DCs results in secretion of both pro-IL-1 (lane 2) and lysosomal cathepsin D (lane 6). Of note, the calcium ionophore triggers
the release of large quantities of IL-1 precursor (20%-40% of the
total pro-IL-1 in the different experiments), but of only minute
amounts of the 29-kd processed form (Figure 5A, lane 2, compare with
secretion induced by T cells in lane 3). This observation indicates
that the [Ca++]i increase itself is not able
to promote an efficient pro-IL-1 processing. Ionomycin also
potentiates the secretion of IL-1 (lane 4) and cathepsin D (lane 8)
induced by alloreactive T cells. Interestingly, secretion of
procathepsin D (lanes 5-8) is only slightly affected by alloreactive T
cells and ionomycin, further supporting that pro-IL-1-containing
lysosomes, rather than endosomes, undergo exocytosis following
interaction with T cells or the [Ca++]i rise.
To evaluate the role of extra- and intracellular Ca++ on
IL-1 and cathepsin D secretion, we compared the effects of EGTA, of
the L-type Ca++ channel agonist Bay K8644, or of
the L-type Ca++ channel blocker nifedipine with those of
thapsigargin, which induces Ca++ release from internal
stores (Figure 5B,C). Removal of extracellular Ca++ by EGTA
prevents the secretion driven by ionomycin treatment (Figure 5B,
EGTA + iono) and decreases that induced by alloreactive T cells
(Figure 5C, EGTA). Furthermore, exposure to Bay K8644 results in
enhancement of secretion, both basal (Figure 5B, BayK) and T-cell
induced (Figure 5C, BayK), whereas nifedipine partially inhibits the
secretion triggered by alloreactive T cells (Figure 5C, NFD). Treatment
of DCs with thapsigargin (Figure 5B, thapsi) induces secretion,
unaffected by EGTA (Figure 5B, EGTA + thapsi), even if at a lesser
extent than Bay K8644. These data suggest that both intra- and
extracellular Ca++ are involved in the secretion elicited
in DCs by the specific interaction with CD8+ T cells;
moreover, they confirm that calcium influx is mediated by L-type
Ca++ channels, that are expressed and functional on
DCs.27 Again, the rate of secretion of procathepsin D is
poorly affected by [Ca++]i modifications,
suggesting that a baseline of procathepsin D release occurs in
LPS-activated DCs and is Ca++ insensitive. To rule out that
secreted IL-1 and cathepsin D derive from T lymphocytes rather
than from DCs, LPS-treated DCs were fixed with glutaraldehyde
before coincubation with alloreactive T cells. In this case, negligible
cathepsin D was found in supernatants at the end of the coculture
(Figure 5C, GA-treated DCs + alloT,  ) and only a small amount
was induced by ionomycin (Figure 5C, GA-treated DCs + alloT,
iono), corresponding to about 20% of that recovered after
ionomycin-stimulation of the same cocultures with nonfixed DCs.
Secreted IL-1 was barely detected or undetectable in all
conditions.
Interaction with CD8+ T cells results in a Ca++ influx in DCs The above observations point to the requirement of a [Ca++]i increase for lysosome exocytosis with release of IL-1 and cathepsin D. We then investigated whether the
interaction between DCs and alloreactive CD8+ T
cells results in a Ca++ influx in DCs. Enriched
CD4+ or CD8+ alloreactive
T cells were loaded with FURA 2-AM and added to DCs,
similarly loaded with the fluorescent probe, and the oscillations in
[Ca++]i were monitored at the single cell
level. Figure 6A shows that the formation
of a contact zone between DCs and alloreactive
CD8+ T lymphocytes is followed by
Ca++ rises in DCs, but not in CD8+ T
cells. Conversely, triggering of DCs by allospecific
CD4+ T cells results in a Ca++
response in T cells, but not in DCs (Figure 6B), in keeping with previous observations.37 Panels C and D in Figure 6 show
the mean of Ca++ responses in 15 DCs interacting with T
cells and confirm that a sustained Ca++ influx is induced
in DCs by CD8+ but not
CD4+ alloreactive T cells.
Polarization of DC endolysosomes toward the interacting CD8+ T cell Ultrastructural analyses show that the interaction between DCs and CD8+ T cells after 5 hours of coculture is associated with recruitment of endolysosomes and mitochondria in the areas of contact among the cells (Figure 7B). Quantitative analyses performed by examining 70 DC-CD8+ T-cell aggregates allowed the calculation that 71% of DCs interacting with CD8+ T lymphocytes displayed lysosome polarization toward the interacting T cells. The lysosome recruitment is already evident after a short coincubation (10-20 minutes) and increases with time (not shown). In contrast, polarization is not observed after binding of CD4+ T cells to DCs (Figure 7A; in 65% of the 70 DC-CD4+ T-cell aggregates examined, CD4+ T lymphocytes interacted with DCs in perinuclear areas displaying no sign of lysosome polarization). No sign of DC morphologic alteration is detectable, confirming the integrity of DCs after culture with T cells. Panels C and D in Figure 7 show a higher magnification of the contact areas among DCs and CD8+ T cells, with details of a tight contact between the plasma membranes of the interacting cells (arrowhead in panel C) and of fusion sites of endolysosomes with the DC plasma membrane (arrows in panels C and D). Immunoelectron microscopy analysis with anticathepsin D antibody (large gold, arrowheads) and anti-IL-1 mAb (small gold, arrows) confirmed the
colocalization of the 2 proteins in the endolysosomes polarized toward
the interacting T cell (Figure 7E and insets). Quantitative analyses
revealed that of 70 T cells interacting with DCs examined, none
displayed intracellular staining for IL-1, confirming the absence of
IL-1 production by alloreactive T cells.
To investigate the mechanism of endolysosome polarization,
T- cell-induced secretion of cathepsin D and IL-1
In this paper we describe a novel mechanism of regulated secretion
in DCs, mediated by Ca++-dependent exocytosis of
endolysosomes, which is induced by CD8+ alloreactive T
cells on interaction with the antigen-presenting cells. This mechanism
of secretion allows the polarized release of IL-1 We have recently shown that IL-1 Unlike in monocytes, where exocytosis of IL-1 Alloreactive CD8+ T cells promote a Ca++ influx
in DCs. Although it is well documented that antigen presentation
results in increases in [Ca++]i in helper T
cells,37 and a low Ca++ response has been
recently shown in 30% of DCs interacting with a CD4+
T-cell clone,41 this is the first report that documents
the ability of CD8+ T lymphocytes to induce
Ca++ mobilization in DCs. Interestingly, a specific
interaction between CD8+ T cells and DCs is required,
because nonspecific CD8+ T cells are able to induce in DCs
neither IL-1 In conclusion, these findings demonstrate the existence of cross-talk
between DCs and CD8+ T lymphocytes. T cells induce the
functional polarization of DCs; in turn, DCs degranulate toward the
triggering T lymphocytes. Although it is conceivable that the
polarized secretion of IL-1
We thank Drs G. Angelini and R. Sitia for critically reading the manuscript, Drs S. Mèresse and A. Santoni and the NCI Biological Resources Branch for the generous gift of antibodies, and the Blood Centers of Gaslini Scientific Institute and Galliera Hospital for providing buffy coats.
Submitted February 27, 2001; accepted May 30, 2001.
Supported in part by grants from Associazione Italiana per le Ricerca sue Cancro and Consiglio Nazionale Ricerche, target project on Biotechnology. C.A. is supported by a fellowship from Fondazione Italiana per le Ricerca sue Cancro.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Anna Rubartelli, National Cancer Research Institute, Largo Rosanna Benzi, 10, 16132 Genova, Italy; e-mail: annarub{at}hp380.ist.unige.it.
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© 2001 by The American Society of Hematology.
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C. Semino, G. Angelini, A. Poggi, and A. Rubartelli NK/iDC interaction results in IL-18 secretion by DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor HMGB1 Blood, July 15, 2005; 106(2): 609 - 616. [Abstract] [Full Text] [PDF]
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C. Andrei, P. Margiocco, A. Poggi, L. V. Lotti, M. R. Torrisi, and A. Rubartelli From The Cover: Phospholipases C and A2 control lysosome-mediated IL-1{beta} secretion: Implications for inflammatory processes PNAS, June 29, 2004; 101(26): 9745 - 9750. [Abstract] [Full Text] [PDF]
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M. M. Al-Alwan, R. S. Liwski, S. M. M. Haeryfar, W. H. Baldridge, D. W. Hoskin, G. Rowden, and K. A. West Cutting Edge: Dendritic Cell Actin Cytoskeletal Polarization during Immunological Synapse Formation Is Highly Antigen-Dependent J. Immunol., November 1, 2003; 171(9): 4479 - 4483. [Abstract] [Full Text] [PDF]
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H. Tapper, W. Furuya, and S. Grinstein Localized Exocytosis of Primary (Lysosomal) Granules During Phagocytosis: Role of Ca2+-Dependent Tyrosine Phosphorylation and Microtubules J. Immunol., May 15, 2002; 168(10): 5287 - 5296. [Abstract] [Full Text] [PDF]
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A. Poggi, R. Carosio, G. M. Spaggiari, C. Fortis, G. Tambussi, G. Dell'Antonio, E. Dal Cin, A. Rubartelli, and M. R. Zocchi NK Cell Activation by Dendritic Cells Is Dependent on LFA-1-Mediated Induction of Calcium-Calmodulin Kinase II: Inhibition by HIV-1 Tat C-Terminal Domain J. Immunol., January 1, 2002; 168(1): 95 - 101. [Abstract] [Full Text] [PDF]
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G. Angelini, S. Gardella, M. Ardy, M. R. Ciriolo, G. Filomeni, G. Di Trapani, F. Clarke, R. Sitia, and A. Rubartelli From the Cover: Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation PNAS, February 5, 2002; 99(3): 1491 - 1496. [Abstract] [Full Text] [PDF]
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and cathepsin D
Top
Abstract
Introduction
,
CD80+, CD86+, HLA-DR+, as
reported.
