Multiple Mechanisms of Reduced Major Histocompatibility Complex
Class II Expression in Endotoxin Tolerance*
Kerstin
Wolk
,
Stefanie
Kunz
,
Nigel
E. A.
Crompton§,
Hans-Dieter
Volk
, and
Robert
Sabat
¶
From the
Institute of Medical Immunology, Medical
School Charité, Humboldt University Berlin, D-10117 Berlin,
Germany and the § Paul Scherrer Institute, CH-5232
Villigen PSI, Switzerland
Received for publication, July 31, 2002, and in revised form, February 21, 2003
 |
ABSTRACT |
Patients after polytrauma, burns, or septic shock
frequently develop a life-threatening immunodeficiency. This state is
associated with specific functional alterations of monocytic cells. We
previously proposed endotoxin tolerance, the monocyte state after acute
response to lipopolysaccharide, as a respective model system. One major feature in both the clinical situation and the in vitro
model is the dramatic down-regulation of monocyte major
histocompatibility complex (MHC) class II surface expression, which is
associated with impaired antigen presentation capacity. This study
focused on the mechanisms behind reduced MHC class II expression in
endotoxin tolerance. Endotoxin priming provoked a decrease of monocyte
intracellular MHC class II. It also led to a reduced expression of the
chaperonic invariant chain and to an inhibited synthesis of the major
lysosomal enzyme for final cleavage of the invariant chain going along
with a relative accumulation of p10. The expression of HLA-DM necessary for loading MHC class II with antigenic peptide was also decreased. Additionally, reduced export of MHC class II 
complexes to the cell surface was observed. The down-regulation of HLA-DR, invariant chain, and HLA-DM was regulated at the mRNA level and may be the consequence of reduced class II transactivator expression observed in
this study. The simultaneous interference at different regulatory levels may explain the uniquely strong and long lasting MHC class II
down-modulating effect of endotoxin priming compared with transforming growth factor-
and interleukin-10. These results not only
contribute to a better understanding of experimental endotoxin
tolerance but may also give rise to new therapeutics for temporary
immunodeficiency and, conversely, for MHC class
II-dependent diseases such as autoimmunity and transplant rejection.
 |
INTRODUCTION |
The exposition of monocytes toward already minor amounts of
endotoxin (LPS)1 provokes a
massive inflammatory response of these cells. However, after initial
LPS response, these cells show a modified reaction toward repeated LPS
exposure. Such monocytes produce only minor amounts of proinflammatory
cytokines, including TNF-
, IL-1
, and IL-12, as well as NO,
whereas the production of IL-1 receptor antagonist and TNF receptor II
was not altered or actually increased (1-6). This monocytic state
after initial LPS priming is therefore designated as endotoxin
tolerance. We previously showed that this altered capacity to respond
to LPS was paralleled by a strong and long lasting down-regulation of
MHC class II expression on these monocytes occurring after a transient
up-regulation of this molecule during LPS priming. The reduced MHC
class II expression was associated with diminished monocyte T-cell
stimulation capacity (7).
Very similar alterations of monocyte function as shown in experimental
endotoxin tolerance are also observed in patients after surgery,
polytrauma, and septic shock. These patients frequently develop a
temporary immunodeficiency, which in its most severe form is referred
to as immunoparalysis and predisposes to often life-threatening
infections (8). The importance of this phenomenon may be reflected by
the fact that in the United States alone more than 200,000 patients die
from sepsis each year (9). Reduced monocyte HLA-DR expression is even
regarded as a diagnostic marker of this temporary immunodeficiency (8).
Unfortunately, there is actually no established causal therapy as yet.
However, the study of the molecular basis of experimental endotoxin
tolerance may help to better understand the mechanisms of this clinical situation.
We therefore focused on the mechanisms of reduced MHC class II
expression in endotoxin tolerance. Expression of antigenic peptide-bearing MHC class II on antigen-presenting cells results from
the coordinated interplay of a number of specific events within the
cell (10-12). Besides the availability of MHC class II-forming
-
and
-chains, various accessory molecules such as invariant chain
CD74, cathepsins, and HLA-DM are essential to ensure MHC class II
protein structure, transport, and peptide loading. This study addressed
these different regulatory levels in human LPS-primed monocytes.
 |
EXPERIMENTAL PROCEDURES |
Preparation and Culture of PBMCs--
Human PBMCs from different
healthy donors were isolated from venous blood and cultured as
previously described (7). To induce the LPS-primed state in monocytes,
PBMCs were cultured in the presence or absence (control group) of 2 ng/ml LPS from Escherichia coli 0127 B8 (Sigma) for 24 h and, if not indicated otherwise, were then extensively washed and
recultured for an additional 24-72 h. For comparison of LPS-primed
cells at 48 h with cytokine-exposed cells, control PBMCs
precultured for 24 h were washed and recultured for the next
24 h in the presence of recombinant TGF-
1 (R & D Systems),
IL-10 (PePro Tech, Rocky Hill, NJ), or a combination of TGF-
1 and
IL-10, each at 10 ng/ml.
To study the effect of blocked cysteine protease activities on monocyte
MHC class II expression, freshly prepared PBMCs were cultured for 4 or
6 h either in the presence of 25 µg/ml E64d (Sigma), in the
respective ethanol concentration (0.08%; v/v) (ethanol control) or
without additives (medium control).
For gene expression analysis, Western blot analysis, and
immunoprecipitation, monocytes were isolated from PBMCs by
CD14-dependent selection using the MACSTM system (Miltenyi
Biotec, Bergisch Gladbach, Germany). As judged by flow cytometric
analysis, isolated fractions contained at least 95% (gene expression
analysis, Western blot analysis) or 90% (immunoprecipitation)
monocytic cells.
Enzymatic Digestion of Cell Surface MHC Molecules--
Papain
treatment of PBMCs was performed as described elsewhere (13) for 2 h using 5 mg/ml papain. Afterward, cells were washed with
phosphate-buffered saline and prepared for flow cytometric analysis of
MHC class I and II expression.
Flow Cytometric Analysis--
For conventional assessment of
monocyte surface antigen expressions and purity, PBMCs were stained
with the following mAbs according to the manufacturer's protocol:
fluorescein isothiocyanate-labeled anti-HLA-A,B,C (G46-2.6),
anti-HLA-DR,DP,DQ (Tü39), anti-CLIP (CerCLIP), mouse IgG1
(MOPC-21), and mouse IgG2a (G155-178), R-phycoerythrin-labeled anti-HLA-DR (L243), anti-HLA-DR (Tü36), mouse IgG2a (X39), and mouse IgG2b (27-35) (all from BD Biosciences, Heidelberg, Germany) and
R-phycoerythrincyanin-labeled anti-CD14 (RMO52; Coulter
Immunotech, Hamburg, Germany).
For assessing the monocyte expression of intracellular
versus extracellular HLA-DR, HLA-DM, and CD74 expression,
cells were incubated for 20 min at 4 °C in the absence (for
detection of HLA-DR, HLA-DM, and CD74) or presence (for detection of
HLA-DR and CD74) of the following unlabeled mAbs: 500 µg/ml
anti-HLA-DR (Tü36), 25 µg/ml anti-HLA-DR (L243), or 500 µg/ml
anti-CD74 (M-B741) (all from BD Biosciences). After extensive washing,
cells were either fixed with paraformaldehyde or fixed and
permeabilized as described previously (14). Afterward, staining was
performed with the following mAbs: fluorescein isothiocyanate-labeled
anti-CD74 (M-B741) and mouse IgG2a (G155-178), R-phycoerythrin-labeled
anti-HLA-DR (Tü36), anti-HLA-DR (L243), anti-HLA-DM (MaP.DM1),
mouse IgG2b (27-35), mouse IgG2a (X39), and mouse IgG1 (MOPC-21) (all
from BD Biosciences) as well as with R-phycoerythrincyanin-labeled anti-CD14 (RMO52; Coulter Immunotech).
To determine the level of monocytic cathepsin S protein expression,
cells were permeabilized as described above and incubated with 2 µg/ml of nonconjugated anti-cathepsin S polyclonal antibodies (C19;
Santa Cruz Biotechnology, Heidelberg, Germany) or with the anti-cathepsin S antibodies blocked with the specific peptide used for
generation of the antibodies (Santa Cruz Biotechnology). Secondary
staining was performed using fluorescein isothiocyanate-labeled donkey
anti-goat IgG (H, L) F(ab')2 fragment (Dianova, Freiberg, Germany).
The flow cytometric analyses were performed by means of a FACSortTM
instrument and CellquestTM software (Becton Dickinson). For each
measurement, 30,000 PBMCs were analyzed, and monocytes were gated based
on their CD14 expression and scatter properties. Data are given as the
difference between the MFI of cells stained with the specific mAb and
the MFI of cells stained with the respective isotype control mAb or, in
case of cathepsin S staining, the peptide-blocked specific antibodies, respectively.
Gene Expression Analysis--
Preparation of total RNA and
reverse transcription of mRNA were performed as described
previously (15). cDNA was analyzed in triplicate assays by TaqManTM
PCR by means of the ABI PrismTM 7700 Sequence Detector System
(Perkin-Elmer Life Sciences) using TaqManTM universal master mix
(Applied Biosystems, Weiterstadt, Germany) and the following sense
primers, antisense primers, and double-labeled oligonucleotide probes:
CTTgg ATgAg CCTCT TCTCA AgC (50 nM), CACCA CgTTC TCTgT
AgTCT CTg (50 nM), FAM-ACTgg gAgTT TgATg CTCCA AgCCC
TC-TAMRA (200 nM) (HLA-DR
); AACCT GAGAC ACCTT AAGAA
CACCA (900 nM), AgTgC CTgCT CATTT CAAAC Ag (300 nM), FAM-TGGTG CATCC AGCTC TCAAA GACCT TC-TAMRA (200 nM) (CD74); CAATG GGAAT GCACT CATAC GAT (300 nM), CTGGG AACTC TCAGG GAACT CA (300 nM), FAM-ATCAC TTCTT CACTG GTCAT GTCTC CCAGG T-TAMRA (200 nM)
(cathepsin S); TGTTT TATGA GGCCC CCAGA T (900 nM), ACAAG
AACCA CACTG ACCCT GATT (300 nM), FAM-TCACA GGAGT CACGT
AGCCT TTCTC TCTCC A-TAMRA (200 nM) (cathepsin L); TCTGC
AGCCC TTGGC TCAT (900 nM), GGGAA CGTCA GAGCG GTTG (300 nM), FAM-CGTAG CCCAC AAGCA ACACC GCAT-TAMRA (cathepsin F);
CgTgg gCATT gTTCT CATCA T (900 nM), gCTgg CATCA AACTC TggTC
T (300 nM), FAM-CAGTC ACCTG AGCAA GGCTT CCGG-TAMRA (200 nM) (HLA-DM
); AgTgC TgggA TTACA AgCgT gA (300 nM), TCggA gAgCA gggTg gAgAa g (300 nM),
FAM-CCACT gCACC gggCC ACAgA gAA-TAMRA (200 nM) (CIITA);
AGTCT GGCTT ATATC CAACA CTTCG (300 nM), GACTT TGCTT TCCTT
GGTCA GG (300 nM), and FAM-TTTCA CCAGC AAGCT TCGAC CTTGA-TAMRA (200 nM) (HPRT). Since preliminary experiments
demonstrated amplification efficiencies in our systems of nearly 1 for
all cDNAs, expressions were calculated relative to those of HPRT as the value 2 to the power of the negative value of the difference between the threshold cycles for the cDNA of interest and HPRT amplification. The absence of significant LPS
priming-dependent variation of monocyte HPRT expression was
verified in 10 experiments using identical amounts of total RNA
(threshold cycles: 26.38 ± 0.26 for controls and 26.33 ± 0.23 for LPS-primed groups) and therefore justifies the use of HPRT as
the housekeeping gene.
Western Blot Analysis--
Monocytes were lysed in buffer
containing 0.5% Triton X-100, 15 mM Tris, 120 mM NaCl, 25 mM KCl, 2 mM
EGTA, 2 mM EDTA, 0.1 mM dithiothreitol,
and protease inhibitors. Insoluble components were removed by
centrifugation. Samples were analyzed directly (for detection of CD74
and CLIP-containing proteins) or after immunoprecipitation (for
detection of MHC class II and CLIP-containing proteins) as follows.
Samples mixed with SDS and sodium deoxycholate (final concentrations of
0.2% (w/v)), NuPage® LDS sample buffer (Invitrogen), and
dithiothreitol (final concentration of 50 mM (w/v)) were
boiled and electrophoresed using NuPAGE® Bis-Tris gel 4-12%
(Invitrogen) and buffer containing 50 mM MES, 50 mM Tris, 3.46 mM SDS, and 20.5 mM
EDTA. Samples were then blotted on nitrocellulose membrane (Invitrogen)
and incubated either with anti-CD74 mAb (M-B741) (BD Bioscience) at 2.5 µg/ml followed by peroxidase-conjugated AffiniPure goat anti-mouse
IgG and IgM (H and L) antibodies (Dianova) at 0.2 µg/ml or with
anti-CLIP polyclonal antibodies (N18) (Santa Cruz Biotechnology) at 1 µg/ml blocked or not with a 400-fold molar excess of peptide used for
generation of the antibodies (Santa Cruz Biotechnology), followed by a
peroxidase-conjugated AffiniPure rabbit anti-goat IgG
F(ab')2 fragment (Dianova) at 0.3 µg/ml, or with
anti-HLA-DR,DP,DQ mAb (Tü39) (BD Biosciences) at 2.5 µg/ml,
followed by peroxidase-conjugated sheep anti-mouse IgG
F(ab')2 fragment (Amersham Biosciences) at a 1:2000
dilution. Detection was finalized using ECL detection reagent (Amersham Biosciences).
Immunoprecipitation--
Monocytes lysed as described
above and blank control containing lysing buffer and CD14 MACSTM beads
also used for monocyte separation (Miltenyi Biotec) were precleared by
incubation with protein G-Sepharose® 4 fast flow (Amersham
Biosciences). The latter was removed than by centrifugation, and
samples were incubated at 4 °C overnight with 1 µg of anti-HLA-DR
mAb (L243) (BD Biosciences) and protein G-Sepharose 4 fast flow
(Amersham Biosciences). The protein G-Sepharose was recovered by
centrifugation and extensively washed in lysing buffer. Elution of
protein G-Sepharose-bound proteins was done after the addition of
NuPage® LDS sample buffer (Invitrogen) and dithiothreitol (final
concentration of 50 mM (w/v)) by heating to 70 °C for 10 min. Eluates were separated from protein G-Sepharose, mixed with SDS
and sodium deoxycholate to final concentrations of 0.2% (w/v), and
analyzed by Western blot analysis.
 |
RESULTS |
One important feature of LPS tolerance, the monocyte state after
acute LPS response, is the down-regulation of monocytic MHC class II
expression (7). In fact, 24-h in vitro priming of human
PBMCs with an already low dose of LPS leads, after a transient up-regulation at 12 h, to strong down-regulation of MHC class II
molecules on monocytes for the following days, as demonstrated by flow
cytometric analysis (see Ref. 7 and Fig.
1A). To compare the effect of
LPS priming with that induced by other known MHC class
II-down-regulating mediators, PBMCs were exposed to TGF-
1 or IL-10
or a combination of both. As demonstrated in Fig. 1B, the
monocyte HLA-DR expression 24 h after the LPS priming period was,
on average, 20.8 ± 0.93% of that of controls and therefore much
more down-regulated than in cells treated for 24 h with IL-10 (61.5 ± 4.81% of controls), TGF-
1 (84.7 ± 4.28% of
controls), or even both cytokines together (48.3 ± 3.45% of
controls). In contrast to HLA-DR expression, the effect of LPS priming
on MHC class I was only marginal (Fig. 1C). Exposition
toward TGF-
1 or IL-10 with or without TGF-
1 had little or no
down-regulating effect on monocyte MHC class I expression,
respectively. LPS priming did not generally reduce monocyte surface
protein expression, since it modestly but reliably up-regulated the
monocytic marker CD14 (Fig. 1D).

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Fig. 1.
Effect of LPS priming on monocyte MHC
expression and comparison with TGF- and IL-10
treatment. A, PBMCs were cultured either with low dose
LPS or without treatment (control) at 0-24 h, and monocyte HLA-DR
expression was analyzed before (0 h), during (12 h), and after (24-72
h) the priming period. Data are given from one representative
experiment as percentage of controls. B-D, PBMCs were
primed with a low dose of LPS at 0-24 h, cultured with TGF- and/or
IL-10 at 24-48 h, or left without treatment (control). Monocyte HLA-DR
(A), HLA-A,B,C (B), and CD14 (C)
expressions were analyzed at 48 h by flow cytometry. MFI values
from three independent experiments are indicated as mean ± S.E.
For HLA-DR assessment, the mAb clone L243 was used.
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The MHC class II analysis described above was performed using the
antibody clone L243, which was used in most studies for detection of a
conformation epitope of the mature HLA-DR-peptide complex. We tested
the expression of other epitopes using additional mAbs. Tü36
detects in any case the HLA-DR even in association with the chaperonic
invariant chain (CD74) that may locate to the cell surface to some
extent (16). Fig. 2A
demonstrates that, compared with nonprimed monocytes, the epitope
recognized by Tü36 was strongly down-regulated in LPS-primed
monocytes as well. This finding suggests that the reduced signal
obtained with the L243 was not primarily due to an increased presence
of HLA-DR-masking CD74 at the cell surface but to a reduction of the
surface HLA-DR level. Additionally, the detection by Tü39, which
even recognizes the isolated
- and
-chains of the different
classical human MHC class II species (HLA-DR, HLA-DP, HLA-DQ), was
similarly reduced on LPS-primed monocytes (Fig. 2A).

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Fig. 2.
Expression of cell surface and intracellular
MHC class II and MHC class II-specific mRNA in LPS-primed
monocytes. PBMCs were primed or not (control) with a low dose of
LPS at 0-24 h. A, at 48 h, monocyte cell surface
expression of different MHC class II epitopes was assessed by flow
cytometry using the mAbs Tü36 and Tü39. MFI values from
three independent experiments are indicated as mean ± S.E.
B, at 48 h, monocyte intracellular HLA-DR 
content was assessed. The Tü36 epitope expression was analyzed by
flow cytometry in permeabilized versus nonpermeabilized
monocytes after cell surface epitope saturation using unlabeled
Tü36. MFI values from three independent experiments are indicated
as mean ± S.E. C and D, before (0 h),
during (12 h), and after (24-72 h) the priming period, monocytes were
isolated and analyzed for HLA-DR-specific mRNA expression by real
time RT-PCR. C, data from one experiment are given as
relative to housekeeping gene expression. D, data from four
experiments are given as percentage of control (mean ± S.E.).
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To assess the cause of down-regulated MHC class II expression in
LPS-primed monocytes, we addressed the different regulatory levels of
MHC class II expression in these cells. The first prerequisite of
HLA-DR expression on cells is the synthesis of this molecule itself. To
assess intracellular MHC class II protein levels, we first tried to
remove the cell surface fraction by papain digestion. In contrast to
the clear reduction of MHC class I, no reduction of monocyte HLA-DR
expression was detected after this treatment (data not shown). We
therefore decided to block cell surface HLA-DR staining by
preincubating cells with an excess of unlabeled Tü36. Thereafter,
cells were permeabilized or not and were stained with a
fluorescence-labeled Tü36. Fig. 2B demonstrates the
results of flow cytometric analysis. In contrast to high levels of
intracellular HLA-DR 
complexes in control monocytes, LPS priming
clearly reduced intracellular HLA-DR staining. Nonpermeabilized cells did not significantly stain with this antibody, demonstrating high
efficacy of extracellular epitope blocking. Similar data were obtained
with Tü39 (data not shown). We further investigated whether the
reduced MHC class II protein expression was associated with reduced MHC
class II mRNA expression. As assessed by quantitative real time
RT-PCR, down-regulation of monocyte HLA-DR
mRNA expression was
observed already during LPS priming (at 12 h), peaking at the end
of the LPS priming period (at 24 h less than 10% of controls) and
recovering the level of untreated controls at 72 h (Fig. 2, C and D). We conclude that monocyte LPS priming
provokes a reduced synthesis of MHC class II.
We further studied the expression of molecules known to participate in
the intracellular pathway of MHC class II. Freshly synthesized MHC
class II
complexes associate in the endoplasmatic reticulum with
CD74, which stabilizes their spatial structure, targets them to the
endosomal compartments with the help of two sorting signals, and
prevents premature peptide loading (10, 11). Monocytes showed only low
expression of surface CD74, being further reduced in LPS-primed cells
(Fig. 3A). Strong
intracellular expression of CD74 was detected in control monocytes. LPS
priming led to clear down-regulation of the intracellular CD74 protein level. As shown in Fig. 3B, this reduced expression was also
observed at the mRNA level (14.3 ± 1.93% of control monocyte
expression), suggesting transcriptional regulation of this
molecule.

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Fig. 3.
Expression of cell surface and intracellular
CD74 and CD74-specific mRNA in LPS-primed monocytes. PBMCs
were primed or not (control) with a low dose of LPS at 0-24 h.
A, at 48 h, monocytes were analyzed by flow cytometry
either directly or after cell surface epitope saturation using
unlabeled anti-CD74 mAb followed or not by cell permeabilization. MFI
values from three independent experiments are indicated as mean ± S.E. B, at 24 h, monocytes were isolated and analyzed
for CD74-specific mRNA expression by real-time RT-PCR. Data from
four experiments are given as relative to housekeeping gene expression
(mean ± S.E.).
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CD74 has been shown to be cleaved in a stepwise fashion by lysosomal
endopeptidases removing the N-terminal endosomal retention signal and
leaving MHC class II groove-binding parts referred to as CLIP. Final
CLIP generation resulting from the cleavage of a 10-kDa CD74 fragment,
p10, seems to be most critical, being realized only by specialized
cysteine endopeptidases. We addressed the role of these enzymes in the
regulation of monocyte MHC class II expression. The exposure of murine
and human B-cell lines to the cysteine protease inhibitor leupeptin has
been shown to induce p10 accumulation and to reduce the transport of
freshly synthesized MHC class II to the cell surface as demonstrated by
pulse-chase experiments (17, 18), indicating a critical role of CLIP
generation for MHC class II cell surface transport in these cells. We
investigated the effect of blocked cysteine protease activity on human
monocyte steady state cell surface expression by exposing PBMCs to
another cysteine protease inhibitor, E64d, for 4 h. As shown in
Fig. 4A, surface MHC class II
expression was reduced to about 60%. This reduction was further
enhanced by extending the incubation time to 6 h (data not shown).
In contrast, no down-regulation of MHC class I was observed in the
presence of E64d, indicating a selective role of cysteine proteases in
the control of expression of monocyte MHC class II.

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Fig. 4.
Role of cysteine protease activity in MHC
class II cell surface expression and impact of LPS priming on major
cysteine protease expression in monocytes. A, PBMCs
were exposed to the cysteine protease inhibitor E64d or the respective
ethanol concentration or were cultured without treatment for 4 h.
The expression of monocyte MHC class II using the mAb Tü39 and
HLA-A,B,C was assessed. Expression relative to ethanol control from
three independent experiments is indicated as mean ± S.E.
B, the mRNA expressions of CLIP-generating cathepsins
were determined in freshly isolated mono- cytes from four donors by real time RT-PCR. Data are given as
relative to housekeeping gene expression. C, PBMCs were
primed or not (control) with a low dose of LPS at 0-24 h, and
monocytes were analyzed for intracellular cathepsin S expression and
expression of cathepsin S-specific mRNA by real time PCR and flow
cytometry, respectively, during (at 12 h) and after (at 24-72 h)
the LPS priming period. Data from one experiment each are given as
percentage of controls. D, PBMCs were primed or not
(control) as in C. At 24 h, monocytes were isolated and
analyzed for cathepsin S-specific mRNA expression by real time
RT-PCR. Data from four experiments are given as percentages of control
(mean ± S.E.). E, PBMCs were primed or not (control)
as in C. At 48 h, monocytes were isolated and analyzed
for expression of CD74 and CD74 fragments (N18 epitope) by Western blot
analysis. The results from one of four experiments are shown.
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The cysteine endopeptidases responsible for the generation of CLIP
(cathepsin S, L, and F) have been investigated (19-22). In myeloid
antigen-presenting cell populations, cathepsin S seems to dominate.
Here we show that human blood monocytes express all (cathepsin S, L,
and F) as assessed by real time RT-PCR (Fig. 4B). Among
them, cathepsin S-specific mRNA was about 100- and 1000-fold more
abundant compared with cathepsin L and F, respectively. In a kinetic
study, we measured the monocyte mRNA and intracellular protein
expression of cathepsin S during and after LPS priming by means of real
time PCR and flow cytometry, respectively. As shown in Fig. 4,
C and D, both mRNA and protein expression
were reduced already during and continuously after LPS priming relative to nonprimed controls. 2 days after LPS priming, cathepsin S mRNA expression recovered, whereas protein expression did not. These data
may suggest an impaired CLIP generation in LPS-primed monocytes. To
investigate whether there is a relative accumulation of CLIP precursor
in LPS-primed monocytes, we performed Western blot analysis using
anti-CD74 and anti-CLIP antibodies. The anti-CLIP antibodies recognized
the CLIP precursors p10 and p22 (Fig. 4E). The specificity of detection was confirmed by the absence of signals when the antibodies were blocked with a 400-fold molar excess of peptide used
for the generation of these antibodies (data not shown). In contrast to
the strong and moderate reduction of CD74 and p22, respectively,
LPS-primed monocytes from all four donors tested showed only hardly
reduced amounts of p10. This relative accumulation of CLIP precursor
agrees with the reduced activity of CLIP-generating enzymes.
MHC class II loading with antigenic peptides is known to take place in
the so-called MHC class II loading compartment with the help of other
accessory molecules such as the nonclassical MHC class II product
HLA-DM. HLA-DM promotes dissociation of CLIP, stabilization of the
empty MHC class II complex, and loading with high affinity peptides
(11). We studied the HLA-DM protein expression in control and
LPS-primed monocytes. No relevant levels were detected on human
monocyte cell surface (Fig.
5A), allowing assessment of
intracellular HLA-DM expression levels without blocking of surface
epitopes. Monocyte LPS priming induced strong down-regulation of this
molecule when compared with controls (Fig. 5A). Reduction of
HLA-DM level could also be demonstrated at the mRNA level
(10.2 ± 1.76% of controls), suggesting that LPS priming
interferes with HLA-DM gene expression in these cells (Fig.
5B). A reduced removal of CLIP from MHC class II 
complexes in LPS-primed monocytes was further supported by the
observation that a higher proportion of cell surface MHC class II was
loaded with CLIP in these cells compared with control cells (data not
shown).

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Fig. 5.
Expression of cell surface and intracellular
HLA-DM and HLA-DM-specific mRNA in LPS-primed monocytes. PBMCs
were primed or not (control) with a low dose of LPS at 0-24 h.
A, at 48 h, monocyte HLA-DM expression was analyzed by
flow cytometry in nonpermeabilized and permeabilized cells. MFI values
from three independent experiments are indicated as mean ± S.E.
B, at 24 h, monocytes were isolated and analyzed for
HLA-DM-specific mRNA expression by real time RT-PCR. Data from four
experiments are given as relative to housekeeping gene expression
(mean ± S.E.).
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The final step in the expression of MHC class II is its export to the
cell surface (10). To investigate whether alterations of the transport
might occur in LPS-primed monocytes, we measured the intracellular and
extracellular expression of the L243 epitope and determined the ratios
of both expressions in control and LPS-primed monocytes from three
donors. As shown in Fig. 6A,
LPS priming increased this ratio, suggesting a relative accumulation of
intracellular MHC class II compared with that in control monocytes.
This accumulation can occur (i) simply because of MHC class II
retention due to diminished degradation of retaining CD74 (caused here
by reduced expression of cathepsin S) and/or (ii) because of direct
alteration of vesicular traffic of mature MHC class II-peptide
complexes as previously shown for IL-10 action (23). Because there are studies demonstrating that the L243 antibody might not fail to detect
MHC class II when associated with CD74 (23), we investigated whether
the L243 was able to detect MHC class II 
complexes associated
with any CD74 fragments in monocytes from our donors used in Fig.
6A. Fig. 6B shows immunoprecipitations with the
L243 antibody in control monocytes, LPS-primed monocytes, and blank cell lysing buffer. Apart from HLA-DR, p22 was co-immunoprecipitated. In monocytes from another donor, the p10 fragment could additionally, though hardly, be seen (data not shown). Again, the specificity of CD74
fragment detection was confirmed by the absence of signals when the
antibodies used were preincubated with a 400-fold molar excess of
blocking peptide (data not shown). These data suggest that in our
monocyte donors the L243 antibody detected not only the mature,
peptide-loaded HLA-DR but also the immature, CD74 fragment-loaded
HLA-DR. Therefore, the relative intracellular accumulation of HLA-DR

complexes in LPS-primed monocytes may be due, at least in part,
to associated, retaining CD74 fragments. Further studies must be
performed to address the extent of direct alteration of vesicular
transport in LPS-primed monocytes.

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Fig. 6.
Export of HLA-DR
 complexes to the cell surface in
LPS-primed monocytes. PBMCs were primed or not (control) with a
low dose of LPS at 0-24 h and analyzed at 48 h. A, the
extracellular and intracellular monocyte expressions of HLA-DR, as
determined by direct cellular staining with the mAb L243 and L243
staining after cell surface-specific epitope saturation and
permeabilization, respectively, were assessed by flow cytometry. The
ratios of intracellular and extracellular expression are indicative for
the export of HLA-DR  complexes to the cell surface. Data from
three independent experiments are given as mean ± S.E.
B, monocytes were isolated, and immunoprecipitation was
performed using the mAb L243 followed by detection of HLA-DR
(Tü39 epitope) and CD74 fragments (N18 epitope) by Western blot
analysis. Results from one out of two experiments are shown.
|
|
Finally, since the expression of HLA-DR, CD74 and HLA-DM is controlled
by CIITA (24), we investigated monocyte CIITA expression before (at
0 h), during (at 12 h), and after LPS priming (at 24, 48, and
72 h) by means of real time RT-PCR. Fig.
7 shows strongly down-regulated CIITA
expression already at 12 h (less than 10% of controls), peaking
at 24 h and being recovered at 72 h. Therefore, reduced
monocytic expression of HLA-DR, CD74, and HLA-DM observed after LPS
priming may be due to reduced CIITA expression in these cells.

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|
Fig. 7.
Expression of CIITA mRNA in LPS-primed
monocytes. PBMCs were primed or not (control) with a low dose of
LPS at 0-24 h. A and B, before (0 h), during (12 h), and after (24-72 h) the priming period, monocytes were isolated
and analyzed for CIITA-specific mRNA expression by real time
RT-PCR. A, data from one experiment are given as relative to
housekeeping gene expression. B, data from four experiments
are given as percentage of control (mean ± S.E.).
|
|
 |
DISCUSSION |
Our data suggest that different steps necessary for cell surface
expression of mature MHC class II are impaired in LPS-primed monocytes.
Besides the strongly reduced MHC class II synthesis, the expressions of
CD74, of the major CLIP-generating cathepsin, and of HLA-DM were
altered, and reduced export of MHC class II to the cell surface was
observed. The essential role of CD74 in MHC class II surface expression
was demonstrated in corresponding knockout mice (25, 26). In these
mice, MHC class II is largely retained in the endoplasmic reticulum due
either to aggregation or to binding of nascent unfolded protein chains.
The importance of cathepsins for MHC class II cell surface expression
is not fully understood. On the one hand, cysteine
protease inhibitors provoke diminished MHC class II cell surface levels
(see Fig. 4A and Refs. 17 and 18), and the increase of
cathepsin S activity during DC maturation was proposed to be
responsible for the massive increase of MHC class II expression (27,
28). On the other hand, cathepsin S deficiency in murine macrophages
and dendritic cells from respective knockout mice show no clear
reduction of cell surface MHC class II levels (19, 20). This
discrepancy may be explained as follows. (i) Newly generated MHC class
II-p10 complexes transiently accumulate upon altered activity of
CLIP-generating enzymes, initially resulting in reduced MHC class II
surface levels. (ii) These complexes also can reach, although delayed,
the cell surface. In case of permanent reduction of cathepsin activity, as in the respective knockout mice, MHC class II surface levels should
be normalized even in the absence of compensating enzymes because of
its regular arrival and half-life at the cell surface. Inborn
HLA-DM-deficient mice show normal levels of cell surface MHC class II
that mainly present CLIP (29), although MHC class II molecules loaded
with CLIP have been postulated to be less stable than those loaded with
selected antigenic peptides. Coming back to our study, we suppose that
even if defects in some isolated steps necessary for MHC class
II-peptide complex expression may be compensated, their simultaneous
inhibition, as presented here in the presence of LPS, results in strong
impairment of peptide presentation.
The effect of LPS priming on monocyte MHC class II expression was even
stronger than that of the prominent immunosuppressive cytokines IL-10
and TGF-
(Fig. 1B). In contrast to LPS, IL-10 has been
excluded from interfering with synthesis of HLA-DR and CD74 (23). Very
recently, Fiebiger et al. (28) also excluded any effect of
IL-10 on constitutive cathepsin S activity using in vitro
generated human dendritic cells. Therefore, the IL-10-induced reduction
of MHC class II cell surface expression in monocytic cells may be due
solely to altered cellular transport of mature MHC class II as proposed
by the group of de Waal Malefyt (23). The same authors also showed that
IL-10 does not affect HLA-DM synthesis and peptide loading of HLA-DR.
TGF-
has been demonstrated to down-regulate MHC class II mRNA
level (30). To our knowledge, no data exist about the effect of TGF-
on other regulation levels of MHC class II expression on monocytic
cells. However, the action of TGF-
via alteration of the class II
transactivator, which also plays a certain role in HLA-DM and CD74 gene
expression, leads us to suppose that TGF-
may also attenuate the
expression of these genes (24, 31). Since IL-10 and TGF-
regulate
MHC class II expression at different levels, they should act in an additive manner. However, the fact that the combination of both cytokines may not affect all considered levels of MHC class II expression regulation might explain their weaker down-regulating effect
compared with LPS priming (Fig. 1B).
Regarding the clinical situation, the results of this study may help
explain an important aspect of the pathomechanism in postinflammatory
immunodeficiency. In fact, although additional mechanisms other than
endotoxinemia may also contribute to the genesis of immunoparalysis,
the functional alterations of monocytes in such patients are very
similar to those of in vitro LPS-primed monocytes.
Conversely, the understanding of the molecular basis of endotoxin
priming-induced impaired MHC class II expression may also be useful for
the development of novel therapeutic approaches in diseases related to
overwhelming MHC class II-dependent immune responses such
as autoimmune diseases and transplant rejection. The fact that
immunoparalytic patients after breaking off immunosuppressive therapy
do not reject transplants demonstrates that such approaches should be
in principle possible. For treatment of immunoparalysis, autoimmunity,
or transplant rejection, the current goal is the identification of
molecular targets that mediate the late, immunosuppressive action of
LPS but not its early, proinflammatory effects. However, we are still
far from realizing such approaches because of the difficulty in
identifying the mechanisms of the late LPS effect as demonstrated
recently by Wysocka et al. (32) with respect to the monocyte
(secondary) LPS response. Since the alterations in endotoxin tolerance
concern different complex monocyte functions such as cytokine
production capacity and antigen presentation, it can be assumed that
also the underlying mechanisms may be complex. Regarding endotoxin
tolerance with respect to reduced MHC class II expression, the
mechanisms should be sought outside the proximal LPS signaling
elements such as toll-like receptor-4, MD2, and MyD88.
 |
ACKNOWLEDGEMENTS |
We acknowledge Dr. W.-D. Döcke
(Schering Co., Berlin) for valuable discussions related to the
manuscript and Dr. A. Vergopoulos (Charité, Berlin) for kind help
with the TaqMan PCR.
 |
FOOTNOTES |
*
This study was supported in part by Deutsche
Forschungsgemeinschaft Grant SFB 421, TP-B2 Volk.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Interdisciplinary
Group of Molecular Immunopathology, Medical School Charité, Humboldt University Berlin, Schumannstr. 20/21, D-10098 Berlin, Germany. Tel.: 49-30-450-518009; Fax: 49-30-450-524932; E-mail: robert.sabat@charite.de.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M207714200
 |
ABBREVIATIONS |
The abbreviations used are:
LPS, lipopolysaccharide;
TNF, tumor necrosis factor;
IL, interleukin;
MHC, major histocompatibility complex;
HLA, human leukocyte antigen;
PBMCs, peripheral blood mononuclear cells;
TGF, transforming growth factor;
E64d, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane
ethyl ester;
mAb, monoclonal antibody;
MFI, mean fluorescence
intensity;
FAM, 6-carboxyfluorescein;
TAMRA, 6-carboxytetramethylrhodamine;
CIITA, class II transactivator;
HPRT, hypoxanthine phosphoribosyltransferase-1;
RT-PCR, PCR on reverse
transcribed mRNA;
CLIP, class II-associated invariant chain
peptides;
MES, 2-(N-morpholino)ethanesulfonic acid.
 |
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