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Prepublished online as a Blood First Edition Paper on January 16, 2003; DOI 10.1182/blood-2002-11-3368.
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Blood, 15 May 2003, Vol. 101, No. 10, pp. 4005-4012
IMMUNOBIOLOGY
 2-Microglobulin as a negative regulator of the
immune system: high concentrations of the protein inhibit in vitro
generation of functional dendritic cells
Jin Xie,
Ying Wang,
Muta E. Freeman III,
Bart Barlogie, and
Qing Yi
From the Myeloma Institute for Research and Therapy,
University of Arkansas for Medical Sciences, Little Rock.
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Abstract |
Two common features in human immunodeficiency virus infection
and acquired immunodeficiency syndrome, rheumatoid arthritis, and
hematologic malignancies including multiple myeloma are elevated serum
levels of  2-microglobulin (2M) and
activation or inhibition of the immune system. We hypothesized that
2M at high concentrations may have a negative impact on
the immune system. In this study, we examined the effects of
2M on monocyte-derived dendritic cells (MoDCs).
The addition of 2M (more than 10 µg/mL) to
the cultures reduced cell yield, inhibited the up-regulation of
surface expression of human histocompatibility leukocyte antigen
(HLA)-ABC, CD1a, and CD80, diminished their ability to activate T
cells, and compromised generation of the type-1 T-cell response induced
in allogeneic mixed-lymphocyte reaction. Compared with control MoDCs,
2M-treated cells produced more interleukin-6 (IL-6),
IL-8, and IL-10. 2M-treated cells expressed
significantly fewer surface CD83, HLA-ABC, costimulatory molecules, and
adhesion molecules and were less potent at stimulating allospecific T
cells after an additional 48-hour culture in the presence of tumor
necrosis factor- and IL-1. During cell culture, 2M
down-regulated the expression of phosphorylated mitogen-activated protein (MAP) kinases, extracellular signal-related kinase (ERK), and
mitogen-induced extracellular kinase (MEK), inhibited nuclear factor- B (NF-B), and activated signal transducer and activator of
transcription-3 (STAT3) in treated cells, all of which are involved in
cell differentiation and proliferation. Thus, our study demonstrates
that 2M at high concentrations retards the generation of
MoDCs, which may involve down-regulation of major histocompatibility
complex class I molecules, inactivation of Raf/MEK/ERK cascade and
NF-B, and activation of STAT3, and it merits further study to
elucidate the underlying mechanisms.
(Blood. 2003;101:4005-4012)
© 2003 by The American Society of Hematology.
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Introduction |
2-Microglobulin (2M)
is an 11.6-kDa nonglycosylated polypeptide composed of 100 amino acids.
It is the invariant chain of the major histocompatibility (MHC) class 1 molecules on the cell surface of all nucleated cells. Its
best-characterized function is to interact with and stabilize the
tertiary structure of the MHC class 1 -chain.1 Because
it is noncovalently associated with the -chain and has no direct
attachment to the cell membrane, 2M on the cell surface
can exchange with free 2M present in serum-containing
medium.2,3 Free 2M is found in body fluids under physiologic conditions as a result of shedding from cell surfaces
or intracellular release. 2M is almost exclusively
catabolized within the kidney; 95% to 100% of circulating
2M is eliminated through glomerular
filtration.4 In healthy persons, the serum concentration
of 2M is usually less than 2 mg/L, and the urinary excretion is less than 400 µg/24 hours.5,6
Two of the common features among diseases such as human
immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS),7,8 autoimmune disorders such as
rheumatoid arthritis9,10 and Sjögren
syndrome,11 and hematologic malignancies including
multiple myeloma, lymphoma, and leukemia,12-15 are
elevated serum levels of 2M and activation or inhibition
of the immune system. In persons infected with HIV, high levels of
serum 2M correlate with the progression to
AIDS,7,8 whereas in the hematologic malignancies, the
levels correlate with poor prognosis.13,14 Thus, based on
the importance of 2M under physiologic and pathologic conditions, we hypothesized that 2M is not only a
surrogate for viral or tumor burdens or a byproduct of immune
activation but, at high concentrations, may have negative effects on
the immune system.
Dendritic cells (DCs) serve as the sentinels of the immune system (for
review, see Banchereau and Steinman16). In their immature
state, DCs reside in peripheral tissues, where they survey for incoming
pathogens. Encounter with pathogens leads to DC activation and
migration to secondary lymphoid organs, where they trigger a specific
T-cell response. DCs are also the most potent antigen-presenting cells
(APCs); they are not only the cells that can stimulate quiescent, naive
CD4+ and CD8+ T cells and B cells and initiate
primary immune responses, they can also induce a strong secondary
immune response with relatively small numbers of DCs and low levels of
antigen. Furthermore, DCs are involved in the polarization of T-cell
responses through secreted cytokines and induction of tolerance through
the deletion of self-reactive thymocytes and the anergy of mature T
cells.16 Given their central role in controlling immunity,
DCs are logical targets for many clinical situations that involve T
cells, such as transplantation, allergy, autoimmune disease, resistance
to infection and to tumors, immunodeficiency, and vaccination. Thus, we
had chosen, in the present study, DCs as the target to test our
hypothesis that 2M may have negative effects on the
immune system. Because sufficient numbers of DCs for in vitro tests can
readily be generated from peripheral blood CD14+ monocytes,
we examined the effects of 2M on monocyte-derived DCs (MoDCs).
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Materials and methods |
Reagents
Human 2M protein purified from urine was
purchased from Sigma (St Louis, MO), and recombinant human
2M was from Calbiochem-Novabiochem (La Jolla, CA).
According to the manufacturers, the purity of the proteins is greater
than 98% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). To ensure their purity, we also analyzed the proteins with
SDS-PAGE, with 4.5% stacking and 15% resolving gel under denaturing
conditions. Then 10 to 20 µg protein was loaded, and no contaminating
proteins were detected (data not shown). Protease inhibitor cocktail,
phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated
monoclonal antibodies against human histocompatibility leukocyte
antigens (HLA)-ABC, HLA-DR, CD1a, CD83, CD80, CD86, CD54, CD40, and
mouse immunoglobulin G1 (IgG1) isotype control were purchased from
PharMingen (San Diego, CA). Anti-CD14 antibody-conjugated microbeads
were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).
Polyclonal antibodies against IB- and IB- were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and
antibodies against mitogen-activated protein (MAP) kinases (MAPKs),
phosphorylated inhibition of nuclear factor-B (IB)-, and
signal transducer and activator of transcription-3 (STAT3) at Tyr705
were from Cell Signaling Technology (Beverly, MA). Recombinant
interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor
(GM-CSF), tumor necrosis factor (TNF)-, and IL-1 were purchased
from R&D Systems (Minneapolis, MN). Tuberculin purified protein
derivative (PPD) was purchased from Statens Serum Institut (Copenhagen,
Denmark). 3[H]-Thymidine and Ficoll-Hypaque were from
Amersham Pharmacia Biotech (Piscataway, NJ).
Generation of monocyte-derived dendritic cells
MoDCs were generated from peripheral blood monocytes using the
standard protocols.17,18 Briefly, peripheral blood
mononuclear cells (PBMCs) were isolated from the blood of healthy
donors using Ficoll-Hypaque gradient centrifugation. In most of the
studies, MoDCs were obtained by incubating PBMCs on a plastic surface
at 37°C in 5% CO2 for 2 hours to allow monocyte
adhesion. After incubation, nonadherent cells were removed, and plates
were vigorously (at least 3 times) washed to eliminate contaminating
cells. In some experiments, monocytes were purified from PBMCs by
positive selection using a MACS column with anti-CD14
antibody-conjugated microbeads. The positively selected fraction
contained more than 95% of CD14+ monocytes. Adherent cells
or purified CD14+ cells were suspended in RPMI 1640 supplement with 10% fetal calf serum, 1 mM glutamine, 100 U/mL
penicillin, 100 µg/mL streptomycin, GM-CSF (100 ng/mL) and IL-4 (100 ng/mL) for 7 days in a humidified incubator at 37°C in 5%
CO2, with the further addition of cytokines on day 3 by
replacing 50% of the medium containing the cytokines. After 7 days of
culture, immature MoDCs were harvested for testing. To induce
maturation, TNF- (10 ng/mL) and IL-1 (10 ng/mL) were added to the
7-day culture, and cells were incubated for another 48 hours. On day 9, mature MoDCs were harvested for testing.
2M treatment of cells
To examine its effects on MoDCs, 2M was added to
the cells at the beginning of culture (day 0). No additional
2M was added on day 3 when 50% of the medium was
replaced or on day 7 when cytokines TNF- and IL-1 were added to
mature MoDCs. In some experiments, 2M was added to
cultures on day 3 to examine whether delayed addition of the protein
could affect the generation of MoDCs.
Flow cytometry analysis
MoDCs harvested on days 7 and 9 were analyzed for their surface
expression of relevant molecules. This was carried out using a
fluorescence-activated cell scan (FACScan) (Becton Dickinson, San Jose,
CA) and was analyzed using the Lysis II program. Briefly, cells were
first washed twice in phosphate-buffered saline (PBS), followed by the
addition of PE- and FITC-conjugated monoclonal antibodies. After
incubation on ice for 30 minutes, the cells were washed twice,
resuspended in PBS, and made ready for analysis. Controls consisted of
cells stained with irrelevant mouse IgG antibodies.
Measurement of cytokines by cytometric bead array analysis
Kits of cytometric bead array analysis for the detection of
cytokines19 including IL-1, IL-6, IL-8, IL-10, IL-12,
TNF- (inflammatory cytokine kit), IL-2, IL-4, IL-5, IL-10,
interferon (IFN)- , and TNF- (T-cell subset cytokine kit)were
purchased from PharMingen, and assay was performed by the Core Facility at the Department of Microbiology and Immunology, University of Arkansas for Medical Sciences. Briefly, supernatants of MoDC or T-cell
culture were collected and kept frozen at  80°C until analysis. When
assayed, the supernatants were mixed with human cytokine-captured beads, and PE-conjugated detection reagent was added and incubated for
3 hours at room temperature. After incubation, the beads were washed 3 times, resuspended in PBS, and ready for flow cytometer analysis.
Mixed-lymphocyte reaction
To examine the capacity of MoDCs to activate allogeneic T cells,
mixed-lymphocyte reaction (MLR) was used. Briefly, purified allogeneic
T cells (5 × 104 cells/100µL/well) were seeded in
96-well U-bottom tissue- culture plates. MoDCs at various numbers were
added and cultured at 37°C in 5% CO2 for 6 days. Sixteen
hours before harvest, 1 µCi (0.037 MBq)
3[H]-thymidine was added to each well. Cells were
harvested, and radioactivity was measured in a -liquid scintillation
analyzer (Packard, Meriden, CT). Results are expressed as the mean of
triplicate cultures.
Presentation of soluble antigen by dendritic cells
To examine the capacity of MoDCs to capture and present soluble
antigen and to activate autologous antigen-specific T cells, assay for
T-cell response to recall antigen PPD was performed using the cells of
healthy blood donors who had been immunized with bacillus
Calmette-Guerin (BCG) vaccines. Results showed a positive T-cell
proliferative response against PPD in vitro. MoDCs were pulsed with 2.5 µg/mL PPD for 2 hours, washed 3 times with PBS, and cultured with
purified autologous T cells for 6 days. T-cell proliferative response
was measured by using overnight incubation with
3[H]-thymidine (1.0 µCi [0.037 MBq]/well), as
described in MLR assay.
Western blot analysis
To detect intracellular signaling associated with
2M treatment, Western blot was used to analyze MAPKs and
nuclear factor-B (NF-B) and STAT3 expression by cultured cells.
To detect MAPKs and IB- and IB-, cells were cultured with
or without 2M, harvested, washed, and lysed with lysis
buffer (50 mM Tris, pH 7.5, 140 mM NaCl, 5 mM EDTA, 5 mM Na3N3, 1%
Triton X-100, 1% NP-40, 1 × protease inhibitor cocktail). For the
determination of phosphorylated MAPKs, IB- and STAT3 (Tyr705),
cells were lysed directly in Laemmli buffer (Sigma). Samples were
centrifuged at 12 000g, and the supernatants were
collected, boiled, and subjected to SDS-PAGE. After transfer to
nitrocellulose membranes and subsequent blocking, membranes were
immunoblotted with the respective antibodies against phosphorylated
MAPKs, IB, or STAT3 and were visualized with alkaline phosphatase-conjugated secondary antibodies; this was followed by
enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA) and
autoradiography. For protein quantification, blots were scanned and
analyzed by spot densitometry using the AlphaImager 2200 Documentation
and Analysis System (Alpha Innotech, San Leandro, CA). Results are
expressed as the average value of pixels enclosed (AVG). AVG is
calculated as the sum of all the pixel values after background
correction divided by area.
Electrophoretic mobility shift assay
Nuclear extracts were prepared from cultured cells using the
Nu-CLEAR extraction kit (N-XTRACT; Sigma). NF-B binding reactions were carried out with 5 µg nuclear proteins and 1 µg NF-B probe according to manual protocol (electrophoretic mobility shift assay [EMSA] gel-shift kits; Panomics, Redwood City, CA). The whole sample
was then loaded on a 6% native polyacrylamide gel in Tris-borate-EDTA (ethylenediaminetetraacetic acid) buffer. After transfer to nylon Biodye-B membrane (Pall, East Hills, NY) and subsequent blocking and
washing, the membranes were visualized using chemiluminescence imaging.
Statistical analysis
Student t test was used to compare various
experimental groups; significance was set at P < .05.
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Results |
2M impaired cell yield and morphology
To examine the effects of 2M on MoDCs, various
concentrations of the protein were added to the cells at the beginning
of the culture. In these experiments, urine-derived 2M
and recombinant 2M were used. During culture, the
differences in cell morphology could be observed as early as day 3;
cells cultured in medium without 2M appeared as mostly
floating, single cells with enlarged, DC-like morphology, whereas cells
cultured with the addition of 2M, especially at more
than 10 µg/mL, remained mostly adherent and smaller and had
monocyte-like shapes. Figure 1A depicts
the morphology of DCs cultured for 7 days in medium only or in medium with the addition of 2M (20 µg/mL). It is obvious that
cells in medium only appeared as large, floating cells with a typical DC-like morphology, whereas cells cultured with 2M,
either urine-derived or recombinant, were smaller and had poorly
differentiated morphology.
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| Figure 1.
Morphologic features and yields of immature MoDCs.
(A) Morphologic features of immature MoDCs harvested on day 7 of
culture with or without the addition of 20 µg/mL urine-derived or
recombinant 2M. Representative fields of differential
interference contrast microscopy (original magnification
× 300) of more than 5 separate experiments are shown. (B) Yields of
immature MoDCs (viable cell counts of big cells only) from day 7 culture with or without the addition of varying concentrations (2.5-40 µg/mL) of urine-derived (U-2M) or recombinant
(R-2M) 2M. Data are expressed as a
percentage of the control. Mean ± SEM from 3 experiments
are shown. *P < .05; **P < .01.
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In addition to the morphologic changes, lower cell yield was also
observed in cultures with 2M, which was determined by
viable cell counts. Figure 1B shows the results of cell recovery from cultures with or without the addition of different concentrations of
2M. It is evident that 2M reduced the
yields of MoDCs, and a significantly lower cell recovery was observed
in cultures with the addition of 10 µg/mL or more 2M
(P < .05 and P < .01). We examined whether
the induction of apoptosis by 2M was responsible for the
low cell yield, using flow cytometry analysis with FITC-conjugated Annexin-V and propidium iodide. No differences were observed between the cultures with or without 2M. In these and the
following studies, PBMCs from 5 healthy blood donors were used to
generate MoDCs.
2M inhibited the up-regulation of surface MHC class
1 and costimulatory molecules
We next examined the surface expression of DC-related molecules by
cultured cells. In these experiments, cells were cultured with or
without the addition of 20 µg/mL 2M for 7 days.
Selection of the concentration of 20 µg/mL 2M for
these and other experiments in this study was based on our preliminary
tests (data not shown), the results depicted in Figure 1B, and the
commonly found serum levels of 2M under pathologic
conditions.7-15 After culture, cells were washed 3 times
and stained with the antibodies. As shown by the representative
histograms of flow cytometry analysis of MoDCs with recombinant
2M on day 7 (Figure 2),
surface expression of CD1a, CD40, CD54, CD80, and HLA-ABC molecules was
significantly (1.5- to 10-fold; P < .05 and
P < 0.01) lower on 2M-treated cells. Interestingly, the expression of CD86 and HLA-DR was increased on
2M-treated cells (1.6- to 2.5-fold;
P < .05). CD14 expression was detected in a small portion
of the cells in culture with 2M but was absent in
control MoDCs, and CD83 levels were low in all the cells (Figure 2).
Prolonged culture of the cells in medium supplemented with GM-CSF and
IL-4 did not restore surface expression of these molecules, because
cells analyzed on days 8 to 10 had similar defects (data not shown).
These results were obtained with urine-derived 2M (data
not shown) and were also reproduced in at least 5 independent
experiments.
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| Figure 2.
Phenotypic features of immature MoDCs.
MoDCs were harvested on day 7 of culture with (shaded histograms) or
without (open histograms, thick lines) the addition of 20 µg/mL
recombinant 2M. Open histograms with thin or broken
lines represent control antibody staining of the cells. Representative
histograms of 5 independent experiments are shown. Gates were
set on the large-size cell populations.
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We also examined whether the delayed addition of 2M
could affect the generation of MoDCs. We added the same concentrations of 2M to the cultures on day 3 and compared cells
recovered from different cultures. No significant difference was
observed in cell yield or morphology between cultures without the
addition of 2M and those with 2M added on
day 3 (data not shown). Flow cytometry analysis revealed no major
changes in cell surface expression of the relevant molecules between
the 2 cultures (data not shown). These results indicate that the
presence of 2M in the first 3 days of culture played an
important role in affecting the generation of MoDCs.
2M altered cytokine secretion profiles of DCs
DCs are the most potent APCs. Not only are they required for the
priming of native CD4+ and CD8+ T cells to
initiate an immune response, they also play an active role in
polarizing the immune response toward type-1 or type-2 T-cell
responses.16 Cytokines secreted by DCs are instrumental to
the process. Therefore, we wanted to examine whether 2M
could also influence the cytokine secretion profile of treated cells. In these experiments, highly purified monocytes were used to generate MoDCs. Supernatants were collected from cultured cells on days 1, 2, 3, and 7, and a flow cytometry-based bead-array analysis was used to
measure the relevant (inflammatory) cytokines. As evident by the
results depicted in Figure 3,
2M treatment (20 µg/mL) significantly up-regulated the
secretion of IL-6 (P < .01; Figure 3A), IL-8
(P < .01; Figure 3B), and IL-10 (P < .05;
Figure 3C), though the concentrations of IL-10 secreted to the culture were lower than those of IL-6 and IL-8. Because of the limitation of
the method, cytokine concentrations higher than 5000 pg/mL cannot be
accurately measured and thus are expressed as more than 5000 pg/mL
(Figure 3B). The concentration of TNF- in culture with the addition
of 2M was dramatically increased on day 1 (P < .01), but it declined to the same low levels found
in control cell cultures from day 2 (Figure 3D). IL-12 was not detected
in the supernatants of the cell cultures. No difference was found in
other cytokines (data not shown). These results were reproduced by
repeated experiments (n = 4) with urine-derived and recombinant 2M.
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| Figure 3.
Cytokine secretion profile of immature MoDCs.
Cultures with ( ) or without ( ) the addition of 20 µg/mL
recombinant 2M. Cytometric bead array analysis was used
to measure the concentrations (pg/mL) of various cytokines in cell
culture medium collected on days 1, 2, 3, and 7. Shown are the results
(mean ± SEM of 4 independent experiments) of cytokines
IL-6 (A), IL-8 (B), IL-10 (C), and TNF- (D).
*P < .05; **P < .01.
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2M treatment compromised MoDC capacity to present
antigens and reduced IL-2 and IFN- production by activated T
cells
Thus far, our results demonstrate that 2M
treatment induced morphologic and phenotypic abnormalities and altered
the cytokine-secretion profile in treated cells. It was likely that the
function of the cellsthat is, antigen presentation and induction of
T-cell activation, which can be assessed by the capacity to activate
allospecific T cells and to present recall antigens to autologous,
antigen-specific T cellswas also compromised. Thus, experiments were
performed to evaluate the function of cultured cells. First, we used
allogeneic MLR to compare MoDCs with or without 2M
treatment in their ability to activate allospecific T cells. Figure
4A shows that cells treated with
2M induced a significantly weaker T-cell response than
those induced by control MoDCs (P < .05). Moreover,
analysis of cytokine secretion by the activated T cells, using the flow
cytometry-based bead-array analysis, revealed that significantly lower
amounts of IL-2 (P < .05) and IFN-
(P < .01) were detected in the supernatants of T cells
stimulated by 2M-treated cells (Figure 4B-C). The amount
of TNF- was also lower, but the difference was not statistically significant. IL-4 and IL-10 levels in the supernatants were low (less
than 10 pg/mL), and no significant difference was noted (data not
shown). No difference was observed in other cytokines (data not shown).
These results indicate that the development of a polarized type 1 T-cell response in the allogeneic MLR culture20,21 was
compromised when 2M-treated cells were used as the APCs.
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| Figure 4.
Antigen-presentation capacity of cultured immature
MoDCs.
(A) 3H-Thymidine incorporation (cpm × 103)
in allogeneic MLR induced by MoDCs cultured with () or without ()
the addition of 20 µg/mL recombinant 2M. (B-C)
Cytokines IFN- and TNF- (B), and IL-2 (C) secreted by
allospecific T cells activated by MoDCs cultured with () or without
() the addition of 20 µg/mL of recombinant 2M.
Values above the bars represent the concentration of the indicated
cytokines. Cytometric bead array analysis was used to measure cytokine
concentrations in the medium of T-cell cultures (containing T cells
[Tc] and DCs at a ratio of 100:1) on day 6. (D)
3H-Thymidine incorporation (cpm × 103) in
autologous T-cell response induced by PPD-pulsed (squares) or unpulsed
(triangles) immature MoDCs cultured with (, ) or without (, )
the addition of 20 µg/mL recombinant 2M. Mean ± SEM of 3 independent experiments are shown.
*P < .05; **P < .01.
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Second, we compared the capacity of MoDCs to present PPD and to
activate autologous specific T cells by using PBMCs from donors who
responded positively to PPD stimulation in vitro. In these experiments,
MoDCs were first pulsed with PPD for 2 hours, washed, and cocultured
with autologous purified CD3+ T cells. As shown in Figure
4D, the specific T-cell response was reduced when
2M-treated cells were used as the APCs
(P < .05). T-cell responses induced by unpulsed MoDCs
were low and no significant difference was found between unpulsed,
2M-treated and control MoDCs. Thus, our results provide
direct evidence that 2M-treated cells have an impaired
antigen-presentation capacity.
2M-induced defects sustained after DC maturation
MoDCs were generated in medium in the presence or absence of
2M at 20 µg/mL. On day 7, cells were washed free of
2M, and recombinant TNF- and IL-1 were added to
the culture to induce DC maturation.17,18 After 48 hours,
cells were harvested and analyzed for their phenotype and function. As
shown in Figure 5A,
2M-treated (in the first 7 days) cells expressed
significantly (1.5- to 10-fold) lower levels of CD83, CD1a, CD40, CD54,
CD80, CD86, and HLA-ABC (P < .05 and
P < .01), even though the expression of these molecules,
except CD54 and CD86, had been up-regulated on
2M-pretreated cells following TNF- and IL-1
treatment. CD1a expression was down-regulated, and that of HLA-DR was
not different. As mentioned previously, most of the abnormalities were
already present in immature 2M-treated cells. We have
also examined the effects of 2M on the maturation of
previously 2M-treated or untreated cells. As depicted in
the representative experiments in Figure 5B, the addition of
2M on day 7 had few, if any, effects on the maturation
of DCs. On the contrary, the presence of this protein from the
beginning of the culture (days 0-7) inhibited the up-regulation of
these molecules, and the further addition of 2M to the
cultures during maturation (days 7-9) had no synergistic effects. As
expected, the secretion of IL-12 was up-regulated in mature MoDCs
(600-1000 pg/mL) but less so in 2M-pretreated cells
(10-50 pg/mL; P < .01; Figure
6A).
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| Figure 5.
Phenotypic features of mature MoDCs harvested on day 9.
(A) Cells were cultured for the first 7 days in medium with (shaded) or
without (open histograms, thick lines) the addition of 20 µg/mL
recombinant 2M, followed by an additional 48-hour
culture in the presence of IL-1 and TNF-. Open histograms with
thin or broken lines represent control antibody staining of the cells.
(B) Dot plots of flow cytometry analysis showing the effects of
2M during maturation (days 7-9) of previously (days 0-7)
2M-treated or untreated DCs. Representative histograms
or dot plots of 5 independent experiments are shown. Gates were set on
the large-size cell populations.
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| Figure 6.
Cytokine secretion and function of mature MoDCs
harvested on day 9.
(A) Secretion of IL-12 by mature MoDCs precultured with () or
without () the addition of 20 µg/mL recombinant 2M
in the first 7 days. (B) 3H-Thymidine incorporation
(cpm × 103) in allogeneic MLR induced by mature MoDCs
precultured with () or without () the addition of 20 µg/mL
recombinant 2M in the first 7 days. Mean ± SEM of
3 independent experiments. *P < .05;
**P < .01.
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Furthermore, 2M-treated cells were also less potent than
control MoDCs at stimulating allospecific T cells
(P < .05) (Figure 6B). These defects remained even after
prolonged maturation (up to 72 hours) with these cytokines or in cells
cultured with CD40 ligand (data not shown). Thus, these results
demonstrate that 2M-induced defects in these cells
sustained even after the maturation of the cells.
2M inhibited MAPK and NF-B activity but activated
STAT3 in treated cells
Our results demonstrate clearly that 2M retarded
the differentiation of monocytes and altered their cytokine expression
profiles. Because MAPKs play a critical role in the regulation of cell
growth and differentiation and NF-B and STAT3 are involved in the
regulation of the expression of or in response to these cytokines, we
examined the levels and activity of these molecules by Western blot
analysis and EMSA. Cell lysates collected on days 1, 3, and 7 of the
cultures were prepared for the analyses. Highly purified monocytes were used in these experiments to generate MoDCs. In this study, the expression of MAPK family membersphosphorylated extracellular signal-related kinase (ERK), p38 MAPK, and SAPK/JNKand of
nonphosphorylated MAPKs, which could serve as controls for protein
loading, was examined. As shown in Figure
7, 2M treatment reduced
the expression of phosphorylated ERK1 or ERK2 (pERK1/2) in the treated
cells. Moreover, the expression of phosphorylated mitogen-induced
extracellular kinase 1 (MEK1) and MEK2 (pMEK1/2), which are the
upstream activators of ERK, was also down-regulated. Levels of
nonphosphorylated ERK and MEK remained stable. No changes were observed
in the expression of other MAPKs, including p38 MAPK or SAPK/JNK (data
not shown). Taken together, these results indicate that the Raf/MEK/ERK
MAP kinase cascade may be involved in 2M-mediated
signaling.
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| Figure 7.
Expression of MAPKs by MoDCs.
Culture in medium with (+2M) or without
(2M) the addition of 20 µg/mL recombinant
2M. (A) Western blots were made with cell lysates
collected on day 1 (D1), day 3 (D3), and day 7 (D7) of the culture.
MAPKs ERK1/2 and MEK1/2 were immunoblotted with specific antibodies.
Phosphorylated (pERK1/2 and pMEK1/2) and nonphosphorylated (ERK1/2 and
MEK1/2) MAPKs were detected. The latter could also serve as control for
protein loading. Representative blots are shown. (B)
Densitometric data (AVG) of pERK1/2 and pMEK1/2 represent the mean ± SEM of 3 independent experiments.
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To detect NF-B activity, we first examined the expression or
activity of its inhibitors, IB- and IB- and phosphorylated IB- (pIB-), because the phosphorylation of serine residues on the IB- proteins (appeared as elevated levels of pIB) by kinases marks them for destruction through the ubiquitination pathway,
thereby allowing activation of the NF-B complex (for review, see
Baldwin22). As shown in Figure
8, compared with those in medium only,
cells cultured in the presence of 2M had higher levels
of pIB- but lower levels of IB- on day 1; this gradually
decreased (pIB-) or increased (IB-), respectively, during
the culture. This suggests that NF-B activity was high in
2M-treated cells on day 1 but declined thereafter. In
contrast, the levels of pIB- were lower and those of IB-
were higher in control cells on day 1. During culture, the levels of
pIB- gradually increased, and those of IB- decreased, in
control cells, suggesting increased activity of NF-B during cell
differentiation induced by IL-4 and GM-CSF. The level of IB-
remained stable. These results correlated very well with NF-B
activity analyzed by EMSA (Figure 8). Collectively, these findings
indicate that, though NF-B activity was low in starting monocytes
and was up-regulated during cell differentiation to MoDCs, its activity
in 2M-treated cells was high initially but decreased
thereafter.
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| Figure 8.
Expression of IB and NF-B and STAT3 by MoDCs
cultured in medium with (+2M) or without (2M) the
addition of 20 µg/mL recombinant 2M.
(A) Western
blots were made with cell lysates collected on day 1 (D1), day 3 (D3),
and day 7 (D7) of culture. IB- and IB-, phosphorylated
IB- (pIB-), STAT3, and phosphorylated STAT3 (pSTAT3) were
immunoblotted with specific antibodies. NF-B activity was analyzed
by EMSA. Representative blots are shown. (B) Densitometric data (AVG)
of pIB- and IB- represent the mean ± SEM of 3 independent experiments.
|
|
For the detection of STAT3 activity, we used an antibody to
detect phosphorylated STAT3 (pSTAT3) at Tyr705, which is an activated form of the molecule. As shown by Figure 8, 2M treatment
up-regulated the expression of pSTAT3 in treated cells and, thus,
enhanced its activity. Little or no STAT3 activity was detected in
cells cultured without the addition of 2M. No
significant difference was observed in the expression of
nonphosphorylated STAT3 protein between cells or in the same cells
during culture.
| |
Discussion |
The present study was designed to investigate whether
2M at high concentrations has a negative impact on the
immune system. As the first step, we examined the effects of
2M on the generation and function of MoDCs because DCs
are the key player in the immune system, and it is now feasible to
obtain substantial numbers of such cells from peripheral blood
monocytes for in vitro experiments. Furthermore, it has been shown that
a proportion of lymph node DCs is derived from monocytes in
vivo.23 PBMCs from healthy blood donors were isolated, and
adherent or purified monocytes were cultured in the presence or absence
of purified human 2M at concentrations ranging from 2.5 µg/mL to 40 µg/mL. These represent the normal levels of
2M in healthy persons5,6 and elevated levels under pathologic conditions.7-15 According to the
data collected in our Institute from more than 1000 myeloma patients, serum 2M can be as high as 80 mg/L (80 µg/mL). One
might expect much higher concentrations of 2M in tumor
sites. Our results demonstrated that, in the presence of high
concentrations (more than 10 µg/mL) of 2M, the in
vitro generation of MoDCs was retarded. This is evident by the low cell
yield, poor morphology, and low expression of surface MHC class 1, CD1a, and costimulatory molecules CD40 and CD80 in treated cells.
Furthermore, these cells had an impaired antigen-presentation capacity.
Compared with control MoDCs, 2M-treated cells secreted
high amounts of IL-6 and IL-8 and the immunosuppressive cytokine IL-10.
They induced significantly weaker allogeneic T-cell activation
and PPD-specific T-cell responses and compromised DC capacity to
mount a type 1 T-cell response (weaker IL-2 and IFN- secretion).
These defects were sustained even after the maturation of MoDCs induced
by culture with TNF- and IL-1 for 48 hours. These results were
seen and reproduced with highly purified human urine-derived
2M and recombinant 2M, indicating that
the effects are indeed mediated by human 2M protein. Hence, our study provides direct evidence to support our hypothesis that 2M has negative effects on the immune system.
High levels of serum 2M are associated with the
progression to AIDS in HIV-infected persons7,8 and with a
poor prognosis in patients with hematologic
malignancies.12-15 Based on the results that DCs
differentiated from monocytes in the presence of high concentrations of
2M had an impaired antigen-presenting capacity and
induced a compromised type 1 T-cell response, our findings may be
compatible with the clinical arena seen in these conditions. The
abnormalities may be attributed to the failure in the up-regulation of
surface expression of MHC class 1 antigen, costimulatory molecule CD80
and adhesion molecule CD54 on the cells, and high amounts of IL-6 and
IL-10 and low amounts of IL-12 secreted by the cells. These cytokines
are known for their role as the negative or positive regulators of type
1 (CD4+ helper T cells [TH1] and
CD8+ cytotoxic T cells) T-cell
differentiation.24,25 In addition, these cytokines,
especially IL-6, are growth and survival factors for tumor cells such
as myeloma cells.26,27 Interestingly, high levels of IL-6
are found in the serum of HIV and multiple myeloma
patients.26-28 Hence, it is conceivable that high levels of 2M might have contributed to disease progression
through its negative effects on the immune system (eg, inhibition of
the generation of virus- or tumor-specific CD4+
TH1 and CD8+ CTL responses) and through
directly promoting tumor cell growth and survival by secreted cytokines.
Although the mechanisms by which 2M-induced
functional retardation in MoDCs are unknown, it is possible that the
effect is mediated through the MHC class 1 molecules. Studies have
shown that MHC class 1 molecules may serve as important
signal-transducing molecules involved in the regulation or fine-tuning
of immune responses (for review, see Skov29). Ligation of
MHC class 1 molecules on T and B cells by mobilized antibodies
triggered signal transduction, which is involved in responses ranging
from anergy and apoptosis to cell proliferation and IL-2
production.29-32 Given that 2M is the
invariant chain of the MHC class 1 molecules, the presence of high
levels of 2M in the culture medium could have affected
the balance and, thus, synthesis of the cellular 2M and
the -chain, as well as the expression and stability of surface MHC
class 1 molecules. Indeed, it is evident that the treatment of cells
with 2M down-regulated surface MHC class 1 expression.
We speculate that high concentrations of exogenous 2M
sent a negative feedback signal to the cells to synthesize less
cellular 2M, which resulted in a reduced number of
assembled MHC class 1 molecules in endoplasmi |
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Abstract
Introduction