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Introduction |
Lipopolysaccharide (LPS, endotoxin) is the major component of the outer membrane of Gram-negative bacteria. The release of LPS by bacteria is considered to be responsible for systemic reactions of severely infected patients
and may cause septic shock associated with high risk for a
lethal outcome. LPS exerts its fatal action not directly, but
stimulates various types of cells, including monocytes/macrophages (1, 2), endothelial cells (3), or granulocytes (4), to
release inflammatory mediators, including IL-1, IL-6, and
TNF-
. In addition, murine B lymphocytes were shown
to respond to LPS by reacting with differential proliferation and antibody production (5). In recent years, it became
evident that LPS is also a potent inducer of human as well
as murine T lymphocyte proliferation and cytokine production (8). We have previously shown that this LPS-induced T cell activation is strongly dependent on direct
cell-to-cell contact of responding T lymphocytes with viable accessory monocytes, indicating receptor-ligand interactions (13). Furthermore, this interaction was MHC unrestricted, but strongly dependent on B7 interaction with
CD28 (14). Moreover, it could be demonstrated by analysis
of the frequency of responding T lymphocytes that LPS is
not active as a mitogen, superantigen, or classical antigen,
indicating a new mechanism of T cell stimulation (14).
When comparing the stimulatory activity of LPS on
PBMCs from adult donors with PBMCs isolated from cord
blood, we made the following unexpected finding: adult
donors could be grouped, with regard to an LPS-inducible
T cell proliferation, into LPS responders and LPS nonresponders (13); however, PBMCs isolated from cord blood
always responded to LPS with a proliferation of T lymphocytes. This phenomenon suggested an important role of a
cell population present in PBMCs from cord blood samples
but rare in PBMCs isolated from adult donors. Only in the
presence of larger amounts of this rare cell population is an
LPS-induced T cell response observed. As described in this
report, this cell type has now been identified as the CD34+
blood hematopoietic stem cell.
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Materials and Methods |
Isolation of Different Cell Populations.
Human PBMCs were isolated from heparinized blood by discontinuous centrifugation
over Ficoll-Hypaque according to the method described by
Böyum (15). CD34+ cells were depleted from PBMCs using
the CD34 Progenitor Cell Isolation kit (Miltenyi Biotech).
Cells were labeled with the hapten-conjugated anti-CD34 mAb
QBEND-10, and immunomagnetic beads were coated with hapten-specific goat anti-mouse (GaM)1 antibodies according to instructions given by the manufacturer.
We were unable to control this depletion procedure by flow
cytometry, since the number of CD34+ cells was always below
the detection limit of this method (in our hands <0.5%). For isolation of CD34+ cells, two subsequent enrichments from PBMCs
were performed, resulting in a purity of CD34+ stem cells between 45 and 85%. In some experiments, lymphocytes were separated from monocytes using a JE-6B elutriator (). The monocyte fraction collected consisted of >95% monocytes, as determined by FACS® analysis after staining with
anti-CD14 (Leu-M3; ). T cells were isolated
from lymphocytes by depletion of CD14+, CD16+, and CD19+
cells using the magnetic activated cell sorting system (MACS®;
Miltenyi Biotech) with the following antibodies: CD14 microbeads, CD16 microbeads, and CD19 microbeads (all Miltenyi
Biotech). These purified T lymphocytes consisted of >98%
CD2+ T lymphocytes (Leu-5b; ) and <0.01%
esterase-positive cells.
Cell Culture.
Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated human serum (HS) and antibiotics in flat-bottomed plates (Nunc) at a final volume of 200 µl.
Cells were stimulated with LPS from Salmonella friedenau (1 µg/ml),
tetanus toxoid (TT, 1 limit of flocculation [Lf]/ml), purified protein derivatives (PPDs) of Mycobacterium tuberculosis (1 µg/ml), or
BCG (4,000 CFU/ml). Cells were cultured for 7 d in a humidified 5% CO2 atmosphere at 37°C. For the last 8 h of stimulation,
cells were labeled with [3H]TdR (2 Ci/mmol, 0.2 µCi/culture)
and harvested on glass filter mats for measurement of incorporated radioactivity.
Induction of CD80 and Immunofluorescence Staining of Cells.
PBMCs (106/ml) were cultured in 24-well plates (1 ml/well;
Nunc) in the presence or absence of LPS (1 µg/ml) in RPMI
1640 supplemented with 10% HS. After 2 d of culture, cells were
collected by careful rubbing to yield all adherent monocytes. Indirect immunofluorescent staining of PBMCs was performed in
ice-cold PBS (containing 0.1% sodium azide) with anti-CD80-
biotin (anti-B7.1, clone BB.1; ) in concentrations as
recommended by the producer. After incubation for 20 min at
4°C, cells were washed by centrifugation on an FCS gradient
(200 g for 10 min). Cells were then incubated for another 20 min
with streptavidin-Red 670. Unbound streptavidin-Red 670 was
again removed by centrifugation over an FCS gradient. Labeled cells were analyzed in a Cytofluorograf (model 50H; Ortho Diagnostic Systems).
IFN-
Production.
PBMCs (106/ml) were cultured in 24-well
plates (1 ml/well; Nunc) in the presence or absence of LPS (1 µg/ml) in RPMI 1640 supplemented with 10% HS. Supernatants
were harvested after 24, 48, or 96 h of culture, and IFN-
production was measured with an ELISA provided by Dr. H. Galatti
(Hoffmann-La Roche, Basel, Switzerland).
Culture Conditions for the Induction of Dendritic Cells.
Monocytes (106/ml) were isolated by counter-flow elutriation and cultured in six-well plates in RPMI 1640 plus 10% HS and GM-CSF (100 U/ml), IL-4 (50 U/ml), and IFN-
(50 U/ml).
Weekly, half of the culture medium was replaced by new medium with cytokines.
 |
Results |
Depletion of CD34+ Blood Stem Cells Prevents LPS-induced
T Cell Proliferation, but Enrichment of CD34+ Blood Stem
Cells Restores the Response of LPS Nonresponders.
In previous investigations, we found that only ~50% of adult
blood donors responded to LPS stimulation by a T cell
proliferation. However, in all PBMCs isolated from cord
blood samples (n > 30), an LPS-induced T cell proliferation could be observed. Thus, we were looking for a very
rare accessory cell population which was significantly enriched in cord blood compared with adult blood. CD34+
cells were likely candidates for this cell population, since they are very rare in adult blood (0.03-0.09%) but present
in significantly larger amounts in cord blood (0.33-1.98%)
(16). Therefore, we depleted CD34+ cells from PBMCs of
adult donors using a CD34 isolation kit and the MACS®
system. These CD34-depleted PBMCs were either stimulated with LPS or the recall antigen PPD of tuberculin.
Furthermore, CD34-enriched cell preparations were added
to CD34-depleted PBMCs and then again stimulated with
LPS or antigens. Table I shows representative results of one
out of seven experiments. Magnetic depletion of CD34+
cells from PBMCs resulted in a clear and almost total loss of the LPS-induced DNA synthesis. The DNA synthesis induced by PPD was not reduced in CD34-depleted cultures, ruling out the possibility that classical APCs were depleted or had lost their accessory capacity during magnetic
depletion procedures. The response to LPS was fully restored or even enhanced by addition of 5% CD34-enriched cells to CD34-depleted PBMCs. These findings were supported by the following control experiments: (a) PBMCs
were labeled with anti-CD34 mAbs and goat anti-mouse
(GaM) microbeads, but not subjected to the MACS® separation columns. This labeling procedure did not affect the LPS-induced T cell proliferation. (b) PBMCs were labeled
with isotype-specific antibodies and after binding to GaM
microbeads were subjected to MACS® separation columns.
These control PBMCs could be stimulated by LPS just as
well as untreated PBMCs. (c) CD34-enriched cells by themselves were stimulated with LPS. As shown in Table I,
these cells did not respond to LPS (or PPD). This finding
excludes the possibility of CD34+ stem cells representing
the proliferating cells after stimulation with LPS.
Next, we investigated whether the addition of CD34-enriched cells to CD34-depleted PBMCs restored an LPS-inducible proliferation of T lymphocytes from LPS nonresponders. As depicted in Table II and demonstrated
previously, DNA synthesis to antigens (PPD, donor 1; TT,
donor 2; BCG, donor 3) was not influenced by depletion or enrichment of CD34+ cells. On the other hand, neither
untreated nor CD34-depleted PBMCs of these LPS nonresponders could be stimulated by LPS. It is noteworthy that
addition of CD34-enriched cells to PBMCs of these donors
clearly resulted in LPS-induced DNA synthesis. Titration experiments showed that as few as 0.5% CD34+ cells were
able to induce significant DNA synthesis after LPS stimulation (data not shown).
CD34+ Blood Stem Cells Are the Responsive Fraction in
Elutriated Monocyte Preparations.
In previous experiments,
we have shown that the stimulation of human T lymphocytes by LPS depends on the presence of monocytes, isolated by counter-flow elutriation. The demonstrated
accessory activity of CD34+ blood stem cells prompted experiments to investigate whether, in fact, CD34+ stem cells
are responsible for the accessory activity of these monocyte
preparations. The monocyte fractions isolated by counter-flow elutriation were depleted from CD34+ stem cells by
the MACS® system. Additionally, CD34+ stem cells were
enriched from monocyte fractions by the MACS® system
using positive selection. Purified T lymphocytes were isolated from PBMCs by counter-flow elutriation and subsequent purification by magnetic depletion of all non-T cells
(CD14+, CD16+, and CD19+ cells). Thereafter, purified T
cells were stimulated with either LPS or PPD in the presence of unseparated monocytes, CD34-depleted monocytes, or CD34-enriched cells. Table III shows the representative results of one of three independent experiments.
Neither LPS nor the recall antigen PPD was able to induce
DNA synthesis in purified T lymphocytes. However, in
the presence of 10% unseparated monocytes, DNA synthesis was observed for both stimuli. Depletion of CD34+ cells
from the elutriated monocyte fractions clearly resulted in
the loss of LPS-induced T cell proliferation, but not of
PPD-induced T cell response. Addition of CD34+ cells not
only restored the response of purified T lymphocytes to
LPS, but resulted in DNA synthesis that was even higher
than in the presence of untreated monocytes. As already
shown for PBMCs, depletion or enrichment of CD34+
cells from elutriated monocytes had no influence on PPD-induced T cell proliferation.
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Table III
Failure of CD34-depleted Monocytes to Exert Accessory Function during Stimulation of T Lymphocytes by LPS
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CD34+ Blood Stem Cells Are Necessary for the Induction of
CD80 on Monocytes after LPS Stimulation.
Previously, we
have reported that stimulation of human T lymphocytes by
LPS is dependent on the interactions of CD80 (B7.1) on
accessory cells and CD28 on T cells (14). We have also
shown that CD80 could be induced on monocytes of LPS
responders, but not on monocytes of LPS nonresponders
after LPS stimulation. To shed light on the role of CD34+
blood stem cells in the expression of CD80, we now compared the CD80 expression on monocytes after LPS stimulation in unseparated PBMCs, CD34-depleted PBMCs,
and CD34-depleted PBMCs plus CD34-enriched cells. As
a control, DNA synthesis was determined. In Table IV, one representative experiment of three independent experiments is shown. CD80 expression on monocytes after LPS
stimulation was clearly dependent on the presence of
CD34+ accessory cells. After LPS stimulation of unseparated PBMCs, CD80 was enhanced on monocytes. Depletion of CD34+ cells resulted in a diminished stimulation of
DNA synthesis as well as reduced CD80 expression after
LPS stimulation. The addition of CD34-enriched cells to
CD34-depleted PBMCs not only restored but even surpassed DNA synthesis as well as CD80 expression after LPS
stimulation compared with PBMC controls.
CD34+ Blood Stem Cells Are Necessary for the Production of
IFN-
.
In previous experiments, we have shown that
LPS is a potent inducer of IFN-
production by human T
lymphocytes (13). In the experiments presented here, we
investigated whether depletion of CD34+ blood stem cells
also influences the production of IFN-
by PBMCs. As
shown in Fig. 1, IFN-
production was clearly reduced in
the absence of CD34+ cells over the total culture period of
4 d. On the other hand, IFN-
production could be fully
restored by adding CD34+ cells. It should be noted that
IFN-
could be detected in PBMCs or CD34
PBMCs
plus CD34+ cells already after 1-2 h of culture (data not
given).

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Fig. 1.
Accessory cell activity of CD34+ blood stem cells during induction of IFN- release in T lymphocytes by LPS. PBMCs (106/ml),
CD34-depleted PBMCs (106/ml), or CD34-depleted PBMCs plus 5%
CD34+ cells were cultured in 24-well plates (1 ml/well) in RPMI 1640 plus 10% HS. Cells were stimulated with LPS (1 µg/ml; black bars); control cultures remained unstimulated (white bars). After 24, 48, or 96 h of
culture, supernatants were harvested and IFN- production was measured
in an ELISA. Data are expressed as mean ± SD of duplicate cultures.
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CD34+ Blood Stem Cells Cannot Be Replaced by Dendritic
Cells Derived from Blood Monocytes.
CD34+ blood stem
cells are described to be a source for the in vitro generation
of dendritic Langerhans cells (17). These dendritic
Langerhans cells were shown to be highly efficient in processing and presenting soluble antigens to autologous T
lymphocytes (20). Thus, we asked whether LPS induces
the generation of dendritic cells from CD34+ blood stem
cells in the system described. Therefore, it was analyzed whether CD34+ stem cells could be replaced by autologous dendritic cells. To obtain dendritic cells, peripheral
blood monocytes were isolated by counter-flow elutriation
and cultured for 2 wk in the presence of GM-CSF, IL-4,
and IFN-
. After this culture period, the cells had the typical morphology of dendritic cells and could be phenotypically characterized by the following surface structures:
CD14, 4.9 ± 2.5%; HLA-DR, 90 ± 7%; CD80, 47.5 ± 6%; and CD86, 53 ± 16%. The accessory capacity of these
dendritic cells was compared with the accessory capacity of
freshly isolated monocytes. As shown in Fig. 2, freshly isolated monocytes induce significantly higher DNA synthesis
after LPS stimulation than dendritic cells. As expected, dendritic cells function as accessory cells after stimulation with TT. From these experiments, we concluded that it is
the CD34+ blood stem cell and not the dendritic cell
which provides accessory capacity in LPS-induced T cell
proliferation.

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Fig. 2.
Accessory activity of monocytes and dendritic cells during
stimulation of T cells by LPS. Purified T lymphocytes (106/ml) were cultured in the presence of 10% freshly isolated autologous monocytes
(white bars) or 10% autologous dendritic cells (black bars) and stimulated
with LPS (S. friedenau, 1 µg/culture) in RPMI 1640 plus 10% HS in a final volume of 200 µl/culture. After 7 d of culture, cells were pulsed with
[3H]TdR (0.2 µCi/culture), then harvested on glass filter mats, and the
radioactivity was measured in a -counter. The results of one of three experiments are given. Data are expressed as mean ± SD of three independent cultures.
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CD34+ Blood Stem Cells Can Be Replaced by KG-1a
Cells.
The purity of our CD34-enriched blood stem cell
preparations varied between 45 and 85%. This raised the
question, whether other cell populations present may contribute to the observed effects in addition to or instead of
CD34+ blood stem cells. Therefore, a myeloid stem cell
line was used to replace accessory cell preparations used.
Indeed, it was found that the hematopoietic stem cell line
KG-1a, derived from an acute myelogenous leukemia (22),
was able to substitute for CD34+ blood stem cell preparations. Purified T lymphocytes were cultured in the presence of
-irradiated KG-1a cells and stimulated with LPS.
Table V shows representative results of eight experiments. It was found that, indeed, T lymphocytes responded to LPS
in the presence of KG-1a cells. Furthermore, controls with
irradiated KG-1a cells showed that the resulting DNA synthesis was not due to proliferating KG-1a cells.
 |
Discussion |
The factors and mechanisms involved in stimulation of
human or murine T lymphocytes by LPS are not yet fully
understood. All features of T cell stimulation indicate a
new, so far unknown mechanism of T cell activation by
LPS: LPS is not a T cell mitogen (low frequency of responding cells), a T cell superantigen (living accessory cells
and CD80-CD28 interactions are necessary), or a classical
T cell antigen (no MHC-restricted interactions between APCs and T lymphocytes) (13, 14). Furthermore, it may be
speculated that LPS, as a lipid antigen, might be presented
(like lipoarabinomannan [LAM]) by CD1 in an MHC-
unrestricted manner. However, monocytes, stem cells, or
KG-1a cells do not express CD1; therefore, this mechanism
of stimulation can be excluded. CD80 expression and IL-12
have been reported to stimulate proliferation and cytokine production in T lymphocytes (23). These data might
suggest that LPS stimulates T lymphocytes by this indirect
mechanism. However, the low frequency of responding T
cells after LPS stimulation (1:1,000; reference 13) excludes
this rather unspecific activation mechanism by CD80 and
IL-12 alone.
The unique and new characteristic feature of T cell stimulation by LPS is underlined by the results presented in this
paper. The necessity of hematopoietic blood stem cells for
activation of T cells by LPS represents a new mechanism of
T cell activation and introduces stem cells as active, potent
cells in inflammatory reactions mediated by T lymphocytes.
Peripheral CD34+ blood stem cells are known to be a
source for autologous and allogenic stem cell transplantation and have been introduced for the treatment of hematological malignancies (26, 27). Furthermore, these cells
have been described to be a source for the in vitro generation of dendritic Langerhans cells by treatment of bone marrow or blood stem cells with GM-CSF and TNF-
(17, 18, 20, 21). These dendritic Langerhans cells were
shown to be highly efficient in processing and presenting
soluble antigen to autologous T lymphocytes (20). However, an accessory function of freshly isolated CD34+ blood
stem cells has not been documented to date. We can only speculate on the mechanisms by which CD34+ blood stem
cells exert their accessory functions. We consider it unlikely that LPS induces the generation of dendritic cells from CD34+ blood stem cells in our system. Time-kinetic
studies concerning the production of IFN-
after LPS
stimulation as shown in Fig. 1 or the expression of CD80
on monocytes after 2 d of culture (Table IV) argue against
this hypothesis. IFN-
could already be detected after a
few hours of stimulation of PBMCs or CD34
PBMCs
plus CD34-enriched cells by LPS (Fig. 1, and our unpublished data). However, as demonstrated by others, for the
generation of dendritic cells, culture periods of several days
are necessary (17). Furthermore, classical dendritic cells
generated from elutriated monocytes by treatment with
IFN-
, IL-4, and GM-CSF were clearly less efficient than
freshly isolated monocyte preparations (Fig. 2).
It has also not been clear whether CD34+ blood stem
cells act directly as accessory cells for the induction of T cell
proliferation or indirectly by influencing the function of
monocytes, which are always present at low numbers in the
CD34-enriched blood stem cell preparations. The requirement for CD80-CD28 interaction for T cell stimulation by
LPS and the requirement of CD34+ stem cells for CD80
expression on monocytes, together with the results obtained with the KG-1a cell line, argue for the latter hypothesis. This question still requires clarification and is presently under further investigation.
In conclusion, the activation of T lymphocytes by LPS
represents a new and unique mechanism of T cell activation differing from that induced by classical mitogens, superantigens, or antigens. The involvement of hematopoietic blood stem cells in this type of activation demonstrated
for the first time a new role of these cells in inflammatory
and immunological events. The significance of these findings for the medication and prevention of diseases (e.g.,
septic shock, bone marrow transplantation, stem cell factor treatment) remains to be investigated.
Address correspondence to Taila Mattern, Research Center Borstel, Center for Medicine and Biosciences,
Parkallee 22, D-23845 Borstel, Germany. Phone: 49-4537-188-448; Fax: 49-4537-188-435; E-mail: ajulmer{at}fz-borstel.de
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