|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3809-3815
IMMUNOBIOLOGY
Secretion of bioactive interleukin-1 by dendritic cells is
modulated by interaction with antigen specific T cells
Stefania Gardella,
Cristina Andrei,
Sara Costigliolo,
Lucia Olcese,
M. Raffaella Zocchi, and
Anna Rubartelli
From the National Institute for Cancer Research, Genova, and the
Laboratory of Tumor Immunology, Scientific Institute San Raffaele,
Milano, Italy.
 |
Abstract |
The role of interleukin-1 (IL-1) as a regulator of the immune
response, although extensively investigated, is still debated. We then
studied the expression of IL-1 by human dendritic cells (DCs), the
professional antigen presenting cells, and its modulation during immune
reactions in vitro. Our results show that, on maturation or tetanus
toxoid presentation to specific CD4+ CD40L+
T lymphocytes, DCs begin to accumulate IL-1 precursor (pro-IL-1) but do not secrete bioactive IL-1. In contrast, interaction with alloreactive T cells results in both stimulation of pro-IL-1 synthesis and secretion of processed isoforms of the cytokine, that
display biologic activity. Both CD4+ and
CD8+ subsets of allospecific T lymphocytes are required:
CD4+ T cells drive the synthesis of pro-IL-1 through
CD40 engagement but have no effects on pro-IL-1 processing;
CD8+ T cells, unable to induce synthesis of pro-IL-1
per se, are responsible for the generation of mature IL-1 by
pro-IL-1-producing DCs. Interleukin-1-converting enzyme (ICE)
inhibitors do not prevent the recovery of IL-1 bioactivity after
allorecognition, indicating that allospecific CD8+ T
cells may induce the release of bioactive IL-1 via mechanism(s) other than ICE activation. Altogether, these findings suggest that
CD4+ and CD8+ T-lymphocyte subsets have
distinct roles in the induction of IL-1 secretion by DCs and support
the hypothesis that IL-1 plays a role in cell-mediated immune responses.
(Blood. 2000;95:3809-3815)
© 2000 by The American Society of Hematology.
| |
Introduction |
Dendritic cells (DCs) are professional
antigen-presenting cells,1,2 able to capture and process
antigens, to migrate into lymphoid organs, and to present antigenic
peptides to naive T cells, inducing the immune response. In most
tissues, DCs are present in the immature state, unable to stimulate T
cells but highly efficient in the uptake of antigens. Bacterial
products, cytokines such as tumor necrosis factor-
(TNF-)3 or the engagement of the DC-surface receptor
CD40 by CD40L expressed by activated T cells,4 induce
maturation, with a rapid decline in the skills to capture antigens and
an increase in the capacity of eliciting a strong T-cell response. DCs
express, constitutively or upon maturation, a number of cytokines that
display biologic activities that are borderline between mediators of
inflammation and modulators of immune response.1 These
include, among others, IL-12,1 TNF-,1 and
IL-18.5 Instead, it is not clear whether DCs express IL-1
and whether this cytokine plays a role during antigen presentation.
IL-1 is the prototypic multifunctional cytokine produced mainly by
activated mononuclear phagocytes.6 Although this soluble
factor was formerly discovered as a costimulator of T lymphocytes, it
soon became clear that its major function is as mediator of host
inflammatory response in natural immunity. However, recent experimental
evidences and clinical trials with IL-1 inhibitors point to a role for
IL-1 in antigen-specific immunity as well as in the pathogenesis of
autoimmune diseases.6 Indeed, even if IL-1 seems
dispensable for mounting an immune response, it appears to regulate the
expansion of different functional T-cell subsets.6-9 In
addition, the inhibition of IL-1 activity, which seems to have little
or no effects on antigen-specific T-cell proliferation,10,11 results in a block of chemokine
production in mixed lymphocyte reaction (MLR).12
Furthermore, a reduction in the severity of a number of immune-mediated
diseases has been observed on treatment with the IL-1 receptor
antagonist.6 Two forms of IL-1 exist, and ; in
humans, the major extracellular form is IL-1, whereas IL-1
remains mainly cell associated.6 Despite its extracellular
localization and function, IL-1 lacks a secretory signal
sequence,13 and is released through a nonclassical pathway
that avoids the ER-Golgi route.14,15 The cytokine is synthesized as a 35-kd precursor, devoid of biologic
activity,6 and is secreted in the mature bioactive form of
17 kd.6,14,15 The IL-1-converting enzyme (ICE) cleaves
pro-IL-1 into the 17-kd form16,17: however, although
ICE is essential for lypopolysaccharide (LPS)-driven IL-1
maturation,18,19 induction of bioactive IL-1 by
different stimuli may occur independently of this
caspase.20,21 Indeed, pro-IL-1 can be cleaved by
cathepsin G,20 chymotrypsin,20 elastase,20 a mast cell chimase,22 different
matrix metalloproteinases,23 and granzyme A24:
These proteases are present in inflammatory fluids and may thus
contribute to extracellular processing of pro-IL-1 with
amplification of the inflammatory response.
To clarify the role of IL-1 in the immune response, we approached
the question of whether the cytokine is produced by immature or mature
DCs and how its release is regulated in the course of interactions with
antigen-specific T cells. Our results indicate that: (i) DCs synthesize
but do not secrete IL-1 on maturation or CD40 triggering; (ii)
secretion of biologically active IL-1 follows an antigen-specific
cell-to-cell contact between DCs and alloreactive T cells; in contrast,
no release of IL-1 occurs on the presentation of a soluble antigen
such as tetanus toxoid to specific CD4+ T cells; and (iii)
in the absence of inflammatory stimuli, both the CD4+ and
the CD8+ subsets of responder T lymphocytes are required.
| |
Materials and methods |
Derivation of dendritic cells from adherent cells
DCs were obtained by culturing adherent cells from peripheral blood
mononuclear cells (PBMCs) from healthy donors for 8 days in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 1 mmol/L
L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Biochrom), 10% heat-inactivated fetal calf serum (FCS) (PAA Labour, Linz, Austria), 40 ng/mL recombinant granulocyte-monocyte
colony-stimulating factor (GM-CSF, Schering-Plough, Milan, Italy), and
1000 U/mL IL-4 as described.25,26 The cell population
obtained was more than 95% CD14 , CD80+,
CD86+, and HLA-DR+, as
reported.25,26 Media were endotoxin free as shown by the Limulus lysate colorimetric assay (PBI, Milan, Italy). LPS (1 µg/mL;
Sigma Chemical Co, St. Louis, MO) or TNF- (50 ng/mL, Genzyme, Cinisello B., Milan, Italy) were added during the last 40 hours of
culture to induce DC maturation.3 Purified T cells were obtained after 2 rounds of plastic adherence, followed by
immunodepletion of CD14+ and HLA-DR+
cells with immunomagnetic beads (Dynal, Milan, Italy).27
Human monocytes enriched by adherence from PBMCs were activated with 10 µg/mL of LPS for 1 hour in RPMI medium, supplemented with 10% FCS as
described.15
Establishment of tetanus toxoid- or dermatophagoides
pteronissinus-specific T-cell lines and mixed lymphocyte reaction
Tetanus toxoid (tetox)-specific or dermatophagoides pteronissinus
(dph)-specific T-cell lines were obtained as described27 by
stimulation of autologous PBMCs with tetox (10 µg/mL, Pasteur-Marieux Connaught, North York, Ontario, Canada) or with dph (5 µg/mL, a kind
gift of Lofarma Allergeni, Milan, Italy), followed by Percoll (Amersham
Italia, Milan, Italy) gradient separation of T-cell blasts after 7 days, culture in 10% T-cell growth factor (Lymphocult, Biotest
Diagnostics Inc, Dreieich, Germany), and weekly restimulation with the
antigen. Allospecific T cells were obtained by coculturing, for 1 week,
purified T cells with allogenic irradiated PBMCs (4000 rad) as
described.27 The percentage of CD8+ and
CD4+ T lymphocytes after 7 days of MLR was evaluated by
FACS analysis. The antigen specificity of dph- or tetox-specific
autologous T cells and of allogeneic T cells was assessed as
described.5,27 CD8+ and CD4+ T
lymphocytes were negatively selected from 7-day MLR by immunodepletion with anti-CD4 or anti-CD8 monoclonal antibodies as
described.27 After removal of CD8+ or
CD4+ cells, respectively, the percentage of
CD4+ or CD8+ T cells in the remaining cell
population (evaluated by FACS analysis) varied from 80% to 95% in the
different experiments.
Culture conditions
DCs, cultured for 6 days in 6-well plates with GM-CSF and IL-4 plus
2 days in the absence or presence of LPS or TNF-, were incubated
with RPMI medium, supplemented with 1% Nutridoma-HU (Boheringer,
Mannheim, Germany) at 37°C for 16 hours with 10 µg/mL of tetox,
followed by 8 hours in the presence or absence of autologous tetox-specific T-cell lines or dph-specific T-cell lines as control at
a 10:1 ratio5,27 or at a different T-cell:DC ratio as indicated.
DCs, prepared as above, were incubated with autologous T cells
activated in a 7-day MLR against an unrelated donor, or allospecific T
cells from 7-day MLR (whole T cells or CD4+ or
CD8+ purified T cells) at a T-cell:DC ratio of
10:1.5,27 Cocultures were carried out for different
periods. When indicated, the ICE inhibitor AcYVAD-CMK (10 µmol/L,
Biochem, Milan, Italy) was added to the cultures. In some experiments,
allospecific T cells were treated with anti-CD40L monoclonal antibody
(10 µg/mL, Ancell, Bayport, MN) for 30 minutes before coculture with DCs.
In other experiments, DCs were challenged with CD40L transfected
cells28 (kind gift of P. Lane, Basel Institute for
Immunology, Basel, Switzerland) at a 1:1 ratio for different periods as indicated.
At the end of the incubations, supernatants were concentrated by 10%
trichloroacetic acid; cells were detached by scraping and lysed in 1%
Triton X-100-containing buffer.29 Aliquots of cell lysates
corresponding to 105 DCs (or 200 µg of proteins, protein
dosage performed with the Bio-Rad kit based on the colorimetric Lowry
method [Bio-Rad, Milan, Italy]) and the correspondent trichloroacetic
acid-concentrated supernatants, were solubilized in sample buffer and
resolved on 12% SDS-polyacrylamide gel electrophoresis (PAGE) under
reducing conditions.29 Gels were electrotransferred onto
nitrocellulose filters (Hybond ECL, Amersham Italia), stained with
Ponceau Red (Sigma) to confirm equal protein loading (not shown), and
destained before blocking overnight with 10% nonfat dry milk in
phosphate-buffered saline (PBS). Filters were hybridized with the
anti-IL-1 monoclonal antibody 3ZD (provided by NCI Biological
Resources Branch, Frederick, MD), followed by a horseradish
peroxidase-conjugated goat antimouse IgG (HRP-GAM, DAKO, Milan, Italy),
or with the goat antihuman ICE antibody (Santa Cruz Biotechnology,
Segrate, Milan, Italy), followed by a horseradish peroxidase-conjugated
rabbit antigoat IgG (DAKO), and developed by ECL-Plus (Amersham),
according to the manufacturer's instructions.
Determination of IL-1 biologic activity
To test the biologic activity of IL-1 secreted by DCs under the
different culture conditions, human umbilical vein endothelial cells
(HUVECs) were grown at confluence in 96-well plates and incubated for
20 hours with the different supernatants, in the presence or absence of
20 pg/mL of recombinant IL-1 receptor antagonist (IRAP, R&D System,
Milan, Italy). Recombinant IL-1 (R&D System) was used as a positive
control at 200 pg/mL. At the end of incubation, the amount of surface
intercellular adhesion molecule-1 (ICAM1) was determined by
enzyme-linked immunosorbent assay (ELISA) with anti-ICAM1 antibodies.
Plates were read with an ELISA reader (Packard) at  = 450 nm.
Results are expressed as optical density (OD) at 450 nm.
| |
Results |
Synthesis of IL-1 by dendritic cells induced by maturational
stimuli
DCs were cultured with GM-CSF/IL-4 for 8 days in the presence or
absence of LPS or TNF- as maturative stimuli for the last 40 hours,3,5 and synthesis and secretion of IL-1 were
investigated in both immature and mature DCs. As shown in Figure
1A, pro-IL-1 (migrating as a 35-kd
band) is absent in DCs and is induced by treatments with LPS or
TNF-. Interestingly, unlike activated monocytes, which mostly
secrete the fully processed 17-kd form of IL-1 (lane 8), mature DCs
release variable amounts of pro-IL-1, but no traces of 17-kd
IL-1 (Figure 1A, lanes 4 to 6). Pro-IL-1 is known to be
biologically inactive6: in keeping with this, we failed to
detect IL-1 activity in the supernatants of TNF-- or
LPS-stimulated DCs (not shown). However, the inactive precursor form of
the pro-IL-1 convertase ICE is present in cell lysates from DCs
either unstimulated or after treatment with LPS or TNF- (Figure 1B,
lanes 1 to 3) in comparable amounts to LPS-stimulated monocytes (lane
4), thus ruling out that the failure of pro-IL-1 processing by
immature and mature DCs is due to the lack of expression of this
enzyme.
View larger version (44K):
[in this window]
[in a new window]
| Fig 1.
Synthesis of pro-IL-1 by DCs is induced by maturative
stimuli.
(A) Cell lysates (lanes 1 to 3, c) and supernatants (lanes 4 to 6, s)
from DCs cultured for 6 days in the presence of GM-CSF and IL-4
followed by 40 hours with GM-CSF/IL-4 alone ( , lanes 1 and 4) or
in the presence of LPS (lanes 2 and 5) or TNF (lanes 3 and 6) were
incubated for 8 hours in serum-free medium supplemented with 1%
nutridoma. Lanes 7 and 8: cell lysates (c) and supernatants (s) from
freshly isolated monocytes stimulated for 3 hours with LPS. Samples
were analyzed by SDS-PAGE, followed by Western blotting with
anti-IL-1 antibodies. (B) Cell lysates from DCs (lanes 1 to 3) or
monocytes (lane 4) untreated (lane 1) or treated with LPS (lanes 2 and
4) or TNF- (lane 3) as indicated. Blots were hybridized with anti
ICE antibodies.
|
|
Activated T cells induce synthesis of pro-IL-1 but not secretion
of bioactive IL-1 in antigen-loaded dendritic cells
To investigate whether the pattern of synthesis and secretion of
IL-1 by DCs is modulated in the course of specific immune responses
to soluble antigens, DCs were pulsed with tetox for 18 hours and
cocultured for 8 hours with autologous antitetox CD4+ T
lymphocytes (proven antigen specific on the basis of their proliferative response to the relevant antigen5,27) at a
T-cell:DC ratio of 10:1, and the presence of pro-IL-1 or mature
IL-1 in DC lysates and supernatants was investigated. Although
overnight incubation with tetox does not induce IL-1 expression
(Figure 2A, lane 2), coculture of
tetox-pulsed DCs with autologous antitetox CD4+ T
lymphocytes results in the induction of pro-IL-1 synthesis, as
indicated by the presence of the specific 35-kd pro-IL-1 band in DC
lysates (lane 3). Of note, the same result is obtained after the
challenge of tetox-loaded DCs with autologous CD4+ T cells
generated against dph (lane 4), whereas no IL-1 synthesis is
observed in the tetox-loaded DCs after coculture with autologous resting T cells (lane 5). These data suggested that the interaction between DCs and activated CD4+ T cells is sufficient to
drive synthesis of IL-1 even in the absence of an antigen-specific
contact. To further investigate this point, DCs pulsed with tetox were
mixed with autologous antitetox or anti-dph CD4+ T cells at
different T-cell:DC ratios (from 0.5:1 to 10:1) and the expression of
IL-1 was investigated after 8 hours. Figure 2 (panels C to E) shows
3 experiments performed with cells obtained from 3 different donors. In
2 cases (panels D and E), no difference in the amount of pro-IL-1
induced by antigen-specific or nonspecific CD4+ T cells is
observed, even at a low T-cell:DC ratio; in 1 donor (panel C),
pro-IL-1 synthesis is induced by tetox-specific CD4+ T
cells at a lower T-cell:DC ratio than by nonspecific T lymphocytes. In
any case, no secretion of mature 17-kd IL-1 is detected, even at a
high-specific CD4+ T-cell:DC ratio (Figure 2B); in
agreement with this, no IL-1 bioactivity is found in supernatants
from the different culture conditions (not shown).
View larger version (5514K):
[in this window]
[in a new window]
| Fig 2.
Activated T cells induce synthesis of pro-IL-1
without secretion.
(A, B) DCs untreated (, lane 1) or loaded with tetox for 16 hours (lanes 2 to 5) were incubated 8 hours alone (lanes 1 and 2) or in the presence of autologous antitetox T lymphocytes
(+Ttetox, lane 3) or autologous antidermatophagoides T lymphocytes
(+Tdph, lane 4) or with autologous resting T cells (+Tresting, lane 5)
at a T-cell:DC ratio of 10:1. At the end of the incubation, cell
lysates (A) and supernatants (B) were analyzed for their content in
IL-1 by Western blotting. (C-E) Autologous specific
(antitetox;  ) or aspecific (anti-dph;  ) T lymphocytes were
coincubated with tetox-loaded DCs for 8 hours at T-cell:DC ratios
varying from 0.5:1 to 10:1. DC intracellular pro-IL-1 was analyzed
by Western blotting and quantitated by densitometry.
|
|
Secretion of bioactive IL-1 by dendritic cells induced by
interaction with allospecific T cells
We then analyzed the modulation of IL-1 synthesis and secretion
by DCs on interaction with alloreactive T cells or with activated nonspecific T cells. Alloreactive T cells were derived from a primary
parallel MLR and were shown to proliferate in response to the allogenic
DCs used in the experiment but not to autologous DCs.5
Nonspecific T cells were activated in an unrelated MLR, and thus were
phenotypically and functionally comparable to alloreactive T cells, but
displayed negligible proliferation to the autologous DCs used in the
experiment.5 As shown in Figure
3A, after 8 hours of incubation with
alloreactive T cells, both induction of pro-IL-1 synthesis (panel
A, lane 2) and secretion of the cytokine (panel B, lane 2) are
observed. Notably, in addition to the precursor form, a 29-kd form and
3 to 4 discrete IL-1 bands with apparent molecular weights ranging
from 12 to 22 kd, which display variable intensity in the different
experiments, are detected in supernatants. Challenging DCs
with nonspecific-activated T lymphocytes also leads to
synthesis of pro-IL-1 (panel A, lane 3); however, differently from
alloreactive T cells, nonspecific T lymphocytes induce secretion of
only a small amount of pro-IL-1 and, in some donors, of the 29-kd
band, with absence of the low-molecular weight bands (panel B, lane 3).
When the same experiment is carried out on LPS-treated DCs, which
constitutively produce and secrete pro-IL-1 (lane 4), similar
results are obtained, although in some cases a stronger induction of
pro-IL-1 synthesis and of IL-1 release by DCs on coculture with
alloreactive T lymphocytes is observed (lane 5). In agreement with the
results of the Western blot analyses, IL-1 secreted after contact of
either untreated or LPS-treated DCs with allospecific T cells is
biologically active, as shown by its property to induce ICAM1
expression on endothelial cells6 (Figure 3C). This ability,
which is comparable to that of rIL-1, is counteracted by
IRAP,6 indicating that the ICAM1 inducing activity present
in the supernatants is indeed IL-1. In contrast, IL-1 bioactivity is
absent from supernatants of DCs, untreated or challenged with
autologous T lymphocytes, even when the 29-kd band is present,
indicating that an antigen-specific interaction is needed to get
secretion of bioactive IL-1.
View larger version (6320K):
[in this window]
[in a new window]
| Fig 3.
Challenge of DCs with alloreactive, but not with
nonspecific T lymphocytes, results in secretion of processed, bioactive
IL-1.
(A, B) DCs, untreated (lanes 1 to 3) or treated with LPS for the last
40 hours (lanes 4 to 6) were challenged with activated allospecific T
lymphocytes (allo T, lanes 2 and 5) or autologous activated T
lymphocytes (auto T, lanes 3 and 6) at a T-cell:DC ratio of 10:1. Cell
lysates (panel A) and supernatants (sups, panel B) were analyzed as for
their content in pro-IL-1 or IL-1 by Western blot. (C)
Bioactivity of secreted IL-1: 100 µL aliquots of supernatants from
the different culture conditions was added to HUVEC and incubated at
37°C for 20 hours. At the end of the incubation, the ICAM1
molecules expressed by HUVEC were quantified by ELISA.
|
|
Kinetics analyses (Figure 4) revealed that,
after incubation with allogenic T cells, both secretion of IL-1 and
ICE activation increase with the time of coculture, as indicated by the
appearance after 1 hour of the ICE-specific 29-kd band that increases
thereafter. The appearance of the low-molecular weight bands is
delayed, as they are easily detectable after 8 hours of coincubation.
View larger version (43K):
[in this window]
[in a new window]
| Fig 4.
Kinetics of IL-1 secretion after contact with
allospecific T cells.
DCs were cocultured with alloreactive T lymphocytes for 1, 3, 6, or 8 hours at a T-cell:DC ratio of 10:1. At the end of each incubation time,
the IL-1 present in the culture supernatants was analyzed by Western
blotting.
|
|
Although the 29-kd and the 17-kd bands are conceivably derived from ICE
processing,6,17,20 it is unclear how the other low-molecular weight forms of secreted IL-1 are generated and whether they are biologically active. To clarify this point, the ICE
inhibitor AcYVAD-CMK was added to the cultures and the pattern of
secreted IL-1 as well as its biologic activity were investigated. As
shown in Figure 5A, ICE inhibition results
in the disappearance of both the 29-kd and 17-kd molecular forms;
however, the IL-1 biologic activity of the supernatants, measured at
different dilutions, is maintained (Figure 5B). These data indicate
that, although ICE undergoes activation on DC-allospecific T-cell
interaction, other convertase(s) are likely to operate on pro-IL-1
giving rise to biologically active IL-1 fragments.
View larger version (2616K):
[in this window]
[in a new window]
| Fig 5.
ICE inhibition prevents the generation of ICE-specific
bands but not the release of IL-1 bioactivity.
(A) Cell lysates (cl, lanes 1 and 2) or supernatants (sups, lanes 3 and
4) from DCs challenged for 8 hours with allogenic T cells at a
T-cell:DC ratio of 10:1, in the absence (, lanes 1 and 3) or
presence (+, lanes 2 and 4) of 100 µmol/L AcYVAD-CMK, were
analyzed as for their content in pro-IL-1 or IL-1 by Western
blot. (B) Bioactivity of secreted IL-1: aliquots of different
dilutions (as indicated) of supernatants of DCs cultured 8 hours alone
() with alloreactive T cells in the absence () or presence ( )
of 100 µmol/L AcYVAD-CMK were added to HUVEC and incubated at
37°C for 20 hours. At the end of the incubation, the ICAM1
molecules expressed by HUVEC were quantified by ELISA.
|
|
Role of CD4+ and CD8+ T
lymphocytes in the modulation of IL-1 synthesis and secretion
The T-cell populations obtained from the different MLR were 50% to
55% CD4+ and 45% to 50% CD8+. To determine
the relevant role of the 2 T-cell subsets in inducing synthesis and/or
secretion of IL-1 by DCs, purified CD4+ or
CD8+ T cells were obtained and cocultured with DCs as
above. As shown in Figure 6A,
CD4+ T lymphocytes (either alloreactive, lanes 1, or nonspecific, not shown) induce pro-IL-1 intracellular
accumulation in immature DCs (upper panel) but not secretion of the
processed forms (bottom panel). At variance, incubation with
alloreactive CD8+ T cells induces neither synthesis nor
secretion of IL-1 by immature DCs (lane 2). However, when DCs are
pretreated with LPS, and hence they are producing pro-IL-1 (Figure
1), interaction with CD8+ alloreactive T cells (lane 4)
results in activation of ICE, as indicated by the appearance of the
29-kd IL-1 band, and secretion of small IL-1 molecular forms,
with generation of bioactive IL-1 (Figure 6, panel B). Again,
IL-1 bioactivity is not prevented by the ICE inhibitor Ac-YVAD-CMK
(not shown), suggesting that additional protease(s), produced or
activated by specific CD8+ T cells, are involved in IL-1
maturation. In contrast, activated CD4+ T cells do not
affect processing and secretion of IL-1 by LPS-treated DCs (Figure
6A, lane 3). It is of note that, as observed for the unfractionated
autologous T cells (Figure 3), autologous CD8+ T
lymphocytes, derived from an MLR against an unrelated stimulator, do
not elicit processing and secretion of bioactive IL-1 by untreated or LPS-treated DCs (Figure 6A, lanes 5 and 6, and Figure 6B), supporting the hypothesis that antigen-specific interactions between CD8+ alloreactive T cells and DCs are needed for the
generation of bioactive IL-1.
View larger version (23K):
[in this window]
[in a new window]
| Fig 6.
Alloreactive CD8+ but not
CD4+ T cells induce generation of bioactive IL-1.
(A) DCs untreated (lanes 1, 2, and 5) or treated for the last 40 hours
with LPS (lanes 3, 4, and 6) were challenged with alloreactive
CD4+ (lanes 1 and 3) or CD8+ (lanes 2 and 4) T
lymphocytes purified from the allospecific T-cell population, or with
autologous CD8+ T lymphocytes purified from an MLR against
an unrelated stimulator (Auto T cells, lanes 5 and 6) at a T-cell:DC
ratio of 10:1. At the end of the coincubation cell lysates (cyt, upper
panel) and supernatants (sec, bottom panel) were analyzed as for their
content in IL-1. (B) Bioactivity of secreted IL-1: 100 µL
aliquots of supernatants from the different culture conditions was
added to HUVEC and incubated at 37°C for 20 hours. At the end of
the incubation, the ICAM1 molecules expressed by HUVEC were quantified
by ELISA.
|
|
CD40 engagement mediates IL-1 synthesis in dendritic cells
The observation that both specific and nonspecific activated
CD4+ T cells are able to induce pro-IL-1 synthesis
suggests that molecular structure(s) expressed by CD4+ T
lymphocytes are responsible for this induction, even in the absence of
an antigen-specific interaction. Triggering of CD40 has been reported
to induce IL-1 expression in macrophages.30 As CD40L is
present on activated CD4+ T cells and CD40-CD40L
interaction has a central role in the immune response,31-32
we investigated whether CD40 engagement results in pro-IL-1
synthesis also in DCs.
Figure 7A shows that culturing DCs with
CD40L-transfected cells at a 1:1 ratio results in a time-dependent
induction of intracellular pro-IL-1. Secreted IL-1 is almost
undetectable in supernatants and IL-1 biologic activity is absent
(not shown). In turn, when activated T lymphocytes are pretreated with
an anti-CD40L antibody and then cultured with DCs, the intracellular
appearance of pro-IL-1 is prevented (Figure 7B). Thus, CD40
triggering is sufficient to induce synthesis of pro-IL-1 but not
secretion of bioactive IL-1.
View larger version (1760K):
[in this window]
[in a new window]
| Fig 7.
CD40-CD40L interaction is responsible for the induction
of IL-1 synthesis by DCs.
(A) DCs were incubated for different periods as indicated with CD40L
transfected cells at a CD40L+ cell:DC ratio of 1:1. At the
end of each incubation time, the intracellular content in pro-IL-1
was analyzed by Western blot and quantified by densitometry. (B) DCs
were incubated for 8 hours alone (lane 1) or with alloreactive T cells
(lanes 2 and 3), that were untreated (lane 2) or pretreated for 30 minutes with anti CD40L antibody, at T-cell:DC ratio 10:1. At the end
of incubation the intracellular content in pro-IL-1 was analyzed by
Western blot.
|
|
| |
Discussion |
In this paper, we show that pro-IL-1 synthesis in DCs
is induced by maturative/inflammatory stimuli, such as LPS, TNF-, or
by coculture with activated-specific or nonspecific CD4+ T
lymphocytes. In all these conditions, however, DCs do not secrete biologically active IL-1. In contrast, when pro-IL-1 synthesis is triggered by either one of these stimuli, the antigen-specific interaction with CD8+ T lymphocytes induces processing and
secretion of mature bioactive IL-1. The IL-1 synthesis driven by
activated CD4+ T cells is mediated at least in part by
CD40-CD40L binding. Thus, similar to what was reported for
monocytes,30 the interaction between CD40L on activated
CD4+ T cells and CD40 on DCs provides a molecular basis for
the induction of pro-IL-1 synthesis. The observation that in one
donor pro-IL-1 expression in DCs was triggered by specific T
lymphocytes at a lower T-cell:DC ratio than by nonspecific T
cells (see Figure 2C), may reflect an improved triggering of CD40 by
CD40L, either quantitative or qualitative, favored by the
antigen-specific binding; alternatively, other antigen-specific
molecular interactions may contribute to the induction of pro-IL-1 synthesis.
The regulation of secretion of IL-1 by DCs differs from that by
monocytes in 2 respects. First, in human monocytes a single stimulus
given by bacterial products is sufficient to drive synthesis, processing and secretion of bioactive IL-1.6,14-15 In
contrast, DCs need at least 2 signals: one, represented by inflammatory stimuli or by CD40L-mediated contact with activated CD4+ T
cells, induces intracellular accumulation of pro-IL-1 and is
not antigen-specific; the other, provided by CD8+ T
lymphocytes, is antigen specific and drives secretion of mature bioactive IL-1. Second, whereas in monocytes processing and
secretion are coupled,6,14,15 in DCs secretion of the
precursor form may precede maturation of the cytokine.
These differences may reflect the different functions of the 2 cell types. Monocytes/macrophages, either resident or recruited, are
mostly involved in inflammation and tissue reactions in natural immunity, in which IL-1 plays a major role.6 At
variance, DCs are specialized in antigen presentation and in the
initiation of the immune response1: in this context, it is
possible that the function of IL-1 is that of amplifying the
inflammatory component of the immune reaction, rather than to act
directly on immunocompetent cells. This may be relevant in autoimmune
diseases with high inflammatory component, such as rheumatoid arthritis.
The processing of the precursor pro-IL-1 molecules induced
by CD8+ T cells is only partially mediated by ICE: indeed,
in addition to the two 29-kd and 17-kd forms generated by
ICE,6,16,17 other low-molecular weight forms are secreted
on contact with allospecific T lymphocytes. Furthermore, although
treatment with the ICE inhibitor Ac-YVAD-CMK prevents, as expected, the
generation of the 29-kd and 17-kd forms, it inhibits neither the
secretion of the additional low-molecular IL-1 forms nor, more
importantly, the recovery of IL-1 bioactivity in the supernatants from
the cocultures. These findings suggest that convertase(s) other than ICE are also involved in the maturation of IL-1 induced by
alloreactive CD8+ T cells. In keeping with this, it has to
be reminded that in ICE / mice, IL-1 activity is
released after stimulation with some proinflammatory
stimuli.20 Furthermore, it has been reported that granzyme
A, a proteolytical enzyme released by cytotoxic T lymphocytes, is able
to generate bioactive IL-1.24 However, in our system,
the release of a soluble enzyme does not seem sufficient, as the
incubation of DCs with autologous T lymphocytes, derived from an MLR
and hence phenotypically and functionally similar to alloreactive T
lymphocytes, fails to induce the secretion of bioactive IL-1.
Thus, the generation and the release of biologically active IL-1
seem to require a cell-to-cell contact between DCs and alloreactive
CD8+ T lymphocytes. The molecular mechanism underlying the
alloreactive T-cell-mediated secretion of mature IL-1 remains to be
elucidated. Understanding this mechanism could lead to the
discovery of novel molecular targets, whereby control of IL-1
production during immune response may be effected.
| |
Acknowledgments |
We thank G. Angelini, A. Poggi, and R. Sitia for critical
comments; P. Lane and Lofarma Allergeni for sharing their reagents; and
the Blood Center of Gaslini Scientific Institute for providing buffy
coats. The 3ZD monoclonal antibody was obtained from the NCI Biological
Resources Branch, FCRDC.
| |
Footnotes |
Submitted August 3, 1999; accepted February 4, 2000.
Supported in part by grants from CNR (Target Project on Biotechnology),
and Ministero Sanità (PF Oncology and special project AIDS). C.A.
is the recipient of a fellowship from "Fondazione Levi-Montalcini."
Reprints: Anna Rubartelli, National Institute for Cancer
Research, Largo Rosanna Benzi, 10, 16132 Genova, Italy; e-mail: annarub{at}hp380.ist.unige.it.
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.
| |
References |
1.
Banchereau J, Steinman R.
Dendritic cells and the control of immunity.
Nature.
1998;392:245-252[Medline]
[Order article via Infotrieve].
2.
Mellman I, Turley SJ, Steinman RM.
Antigen processing for amateurs and professionals.
Trends Cell Biol.
1998;8:231-237[Medline]
[Order article via Infotrieve].
3.
Sallusto F, Lanzavecchia A.
Dendritic cells use macropinocytosis and the mannose receptor to concentrate antigen to the MHC class II compartment: downregulation by cytokines and bacterial products.
J Exp Med.
1995;182:389-400[Abstract/Free Full Text].
4.
Caux C, Massacrier C, Vanbervliet B, et al.
Activation of human dendritic cells through CD40 cross-linking.
J Exp Med.
1994;180:1263-1272[Abstract/Free Full Text].
5.
Gardella S, Andrei C, Costigliolo S, Poggi A, Zocchi MR, Rubartelli A.
Interleukin-18 synthesis and secretion by dendritic cells are modulated by interaction with antigen specific T cells.
J Leuk Biol.
1999;66:237-241[Abstract].
6.
Dinarello CA.
Biological basis for interleukin-1 in disease.
Blood.
1996;87:2095-2147[Abstract/Free Full Text].
7.
Greenbaum LA, Horowitz JB, Woods A, Pasqualini T, Reich E-P, Bottomly K.
Autocrine growth of CD4+ T cells: differential effects of IL-1 on helper and inflammatory T cells.
J Immunol.
1988;140:1555-1560[Abstract].
8.
Bhardwaj N, Lau LL, Friedman SM, Crow MK, Steinman RM.
Interleukin-1 production during accessory cell-dependent mitogenesis of T lymphocytes.
J Exp Med.
1989;169:1121-1136[Abstract/Free Full Text].
9.
Satoskar AR, Okano M, Connaughton S, Raisanen-Sokolwski A, David JR, Labow M.
Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice.
Eur J Immunol.
1998;28:2066-2074[Medline]
[Order article via Infotrieve].
10.
Nicod LP, el Habre F, Dayer JM.
Natural and recombinant interleukin-1 receptor antagonist does not inhibit human T-cell proliferation induced by mitogens, soluble antigens or allogenic determinants.
Cytokine.
1998;41:29-35.
11.
Faherty DA, Claudy V, Plocinski JM, et al.
Failure of IL-1 receptor antagonist and monoclonal anti IL-1 receptor antibody to inhibit antigen specific immune responses in vivo.
J Immunol.
1992;148:766-771[Abstract].
12.
Lukacs NW, Kunkel SL, Burdick MD, Lincoln PM, Strieter R.
Interleukin-1 receptor antagonist blocks chemokine production in the mixed lymphocyte reaction.
Blood.
1993;82:3668-3674[Abstract/Free Full Text].
13.
Rubartelli A, Sitia R.
Secretion of mammalian proteins that lack a signal sequence. In:
Kuchler K,Rubartelli A,Holland B, eds.
Unusual Secretory Pathways: From Bacteria to Man. Austin, TX: RG Landes Company; 1997:87-104.
14.
Rubartelli A, Cozzolino F, Talio M, Sitia R.
A novel secretory pathway for Interleukin-1, a protein lacking a signal sequence.
EMBO J.
1990;9:1503-1510[Medline]
[Order article via Infotrieve].
15.
Andrei C, Dazzi C, Lotti L, Torrisi MR, Chimini G, Rubartelli A.
The secretory route of the leaderless protein IL-1 involves exocytosis of endolysosome-related vesicles.
Mol Biol Cell.
1999;10:1463-1475[Abstract/Free Full Text].
16.
Thornberry NA, Bull HG, Calaycay JR, et al.
A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.
Nature.
1992;356:768-774[Medline]
[Order article via Infotrieve].
17.
Cerretti DP, Kozlosky CJ, Mosley B, et al.
Molecular cloning of the Interleukin-1 converting enzyme.
Science.
1992;256:97-100[Abstract/Free Full Text].
18.
Li P, Allen H, Banerjee S, et al.
Mice deficient in IL-1 converting enzyme are defective in production of mature IL-1 and resistant to endotoxic shock.
Cell.
1995;80:401-411[Medline]
[Order article via Infotrieve].
19.
Kuida K, Lippke JA, Ku G, et al.
Altered cytokine export and apoptosis in mice deficient in interleukin-1 converting enzyme.
Science.
1995;267:2000-2003[Abstract/Free Full Text].
20.
Fantuzzi G, Ku G, Harding MW, et al.
Response to local inflammation of IL-1-converting enzyme-deficient mice.
J Immunol.
1997;158:1818-1824[Abstract].
21.
Miwa K, Asano M, Horai R, Iwakura Y, Nagata S, Suda T.
Caspase 1-independent IL-1 release and inflammation induced by the apoptosis inducer Fas ligand.
Nat Med.
1998;4:1287-1292[Medline]
[Order article via Infotrieve].
22.
Mizutani H, Schechter N, Lazarus G, Black RA, Kupper TS.
Rapid and specific conversion of precursor interleukin-1 (IL-1) to an active IL-1 species by human mast cell chimase.
J Exp Med.
1991;174:821-825[Abstract/Free Full Text].
23.
Schoenbeck U, Mach F, Libby P.
Generation of biologically active IL-1 by matrix metalloproteases: a novel caspase-I independent pathway of IL-1 processing.
J Immunol.
1998;161:3340-3346[Abstract/Free Full Text].
24.
Irmler M, Hertig S, MacDonald HR, et al.
Granzyme A is an interleukin-1 converting enzyme.
J Exp Med.
1995;181:1917-1922[Abstract/Free Full Text].
25.
Sallusto F, Lanzavecchia A.
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and down-regulated by tumor necrosis factor .
J Exp Med.
1994;179:1109-1118[Abstract/Free Full Text].
26.
Rubartelli A, Poggi A, Zocchi MR.
The selective engulfment of apoptotic bodies by dendritic cells is mediated by the v3 integrin and requires intracellular and extracellular calcium.
Eur J Immunol.
1997;27:1893-1900[Medline]
[Order article via Infotrieve].
27.
Ferrero E, Bondanza A, Leone BE, Manici S, Poggi A, Zocchi MR.
CD14+CD34+ peripheral blood mononuclear cells migrate across endothelium and give rise to immunostimulatory dendritic cells.
J Immunol.
1998;160:2675-2683[Abstract/Free Full Text].
28.
Lane P, Traunecker A, Hubele S, Inui S, Lanzavecchia A, Gray D.
Activated human T cells express a ligand for the human B cell-associated antigen CD40 which participates in T cell-dependent activation of B lymphocytes.
Eur J Immunol.
1992;22:2573-2578[Medline]
[Order article via Infotrieve].
29.
Hamon Y, Luciani MF, Becq F, Verrier B, Rubartelli A, Chimini G.
Interleukin-1 secretion is impaired by inhibitors of the ATP binding cassette transporter, ABC1.
Blood.
1997;90:2911-2915[Abstract/Free Full Text].
30.
Wagner DH, Stout RD Jr, Suttles J.
Role of CD40-CD40L interaction in CD4+ T cell contact-dependent activation of monocyte interleukin-1 synthesis.
Eur J Immunol.
1994;24:3148-3154[Medline]
[Order article via Infotrieve].
31.
Grewal IS, Flavell RA.
A central role of CD40 ligand in the regulation of CD4+ T cell responses.
Immunol Today.
1996;17:410-414[Medline]
[Order article via Infotrieve].
32.
van Kooten C, Banchereau J.
Functions of CD40 on B cells, dendritic cells and other cells.
Curr Opin Immunol.
1997;9:330-337[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
M. Ceppi, P. M. Pereira, I. Dunand-Sauthier, E. Barras, W. Reith, M. A. Santos, and P. Pierre
MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells
PNAS,
February 24, 2009;
106(8):
2735 - 2740.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Carta, S. Tassi, C. Semino, G. Fossati, P. Mascagni, C. A. Dinarello, and A. Rubartelli
Histone deacetylase inhibitors prevent exocytosis of interleukin-1beta-containing secretory lysosomes: role of microtubules
Blood,
September 1, 2006;
108(5):
1618 - 1626.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
T. Luft, M. Jefford, P. Luetjens, H. Hochrein, K.-A. Masterman, C. Maliszewski, K. Shortman, J. Cebon, and E. Maraskovsky
IL-1{beta} Enhances CD40 Ligand-Mediated Cytokine Secretion by Human Dendritic Cells (DC): A Mechanism for T Cell-Independent DC Activation
J. Immunol.,
January 15, 2002;
168(2):
713 - 722.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Gardella, C. Andrei, L. V. Lotti, A. Poggi, M. R. Torrisi, M. R. Zocchi, and A. Rubartelli
CD8+ T lymphocytes induce polarized exocytosis of secretory lysosomes by dendritic cells with release of interleukin-1{beta} and cathepsin D
Blood,
October 1, 2001;
98(7):
2152 - 2159.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
Top
Abstract
Introduction