Full text of DOI:10.1111/j.1348-0421.2002.tb02686.x

Microbiol. Immunol., 46(3), 195―205, 2002
IL-10 Producing CD14low Monocytes Inhibit
Lymphocyte-Dependent Activation of Intestinal
Epithelial Cells by Commensal Bacteria
Dirk Haller1, 4, Patrik Serrant1, Genneviève Peruisseau1, Christiane Bode2,
Walter P. Hammes3, Eduardo Schiffrin1, and Stephanie Blum*, 1
1Department of Immunology, Nestlé Research Centre, Vers-chez-les-Blanc, Ch-1000 Lausanne 26, Switzerland, 2Institute of
Biological Chemistry and Nutrition Science, Hohenheim University, Garbenstrasse 28, D-70599 Stuttgart, Germany,
4
3Institute of Food Technology, Hohenheim University, Garbenstrasse 28, D-70599 Stuttgart, Germany, and Center of Intesti-
nal Biology and Disease, University of North Carolina at Chapell Hill, U.S.A.
Received October 26, 2001; in revised form, December 12, 2001. Accepted December 21, 2001
Abstract: Intestinal epithelial cell (IEC) activation by non-pathogenic, commensal bacteria was demonstrated
to require the presence of immunocompetent cells. In this study, HT-29 and CaCO-2 transwell cultures,
＋
＋
＋
reconstituted with CD4 and CD8 T cells, CD19 B cells and CD14high monocytes, were challenged with non-
pathogenic Gram negative Escherichia coli and Gram positive lactobacilli. Cytokine expression was
analysed by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent
assays (ELISA). Expression of tumour necrosis factor alpha (TNF-ꢀ) and interleukin (IL)-8 mRNA in E.
＋
coli or L. sakei challenged IEC was promoted by lymphocyte populations predominantly CD4 T cells, while
monocytes failed to mediate an inflammatory cytokine response. The monocyte phenotype and function were
further characterised by flow cytometry and mixed lymphocyte reaction (MLR). During the co-culture with
IEC and bacterial stimulated IEC, CD14high peripheral blood monocytes acquired a CD14low CD16low phe-
notype with reduced expression co-stimulatory (CD80, CD86, CD58) cell surface molecules. Immunosup-
pressive functions of IEC conditioned CD14low monocytes were demonstrated by the predominant secretion
of IL-10 and IL-1Ra and their reduced potential to trigger an allogeneic lymphocyte response. In conclu-
sion, IEC contribute to the development of CD14low CD16low monocytes with immunosuppressive function
and antagonised a lymphocyte-mediated activation of the intestinal epithelium in response to intestinal and
food derived bacteria. These results strengthen the hypothesis that the gut epithelium constitutes an
important functional element in the regulation of mucosal immune homeostasis towards commensal bac-
teria.
Key words: Intestinal epithelial cells, CaCO-2 and HT-29 transwell co-cultures, TNF-ꢀ, IL-8, IL-10,
CD14low CD16low monocytes, Lactobacilli, Commensal bacteria
The indigenous microflora harbouring the human
intestine is in close proximity to a large number of spe-
cialised, lymphoid and accessory cells present through-
out the lamina propria, the follicles of the gut-associated-
lymphoid tissue (GALT), and intraepithelial lympho-
cytes (27). The maintenance of local homeostasis in the
intestinal mucosa to luminal bacterial antigens requires
a fine tuning of immunological processes, particularly
those involving T cell activation (31, 41). The lack of
responsiveness or oral tolerance towards the autologous
intestinal microflora is a physiological feature that, if
impaired, may lead to intestinal inflammation (12, 13, 15,
18
―
20). It has been suggested that the hyporeactivity of
lamina propria T cells to stimulation through the T cell
receptor/CD3 complex could be one mechanism of the
mucosal compartment securing immunoregulation at
the antigen-specific level (43, 45, 58). This appears, at
least in part, to be due to impaired signal transduction,
probably mediated by soluble factors produced by intesti-
nal epithelial cells (IEC) (11). Resident intestinal mono-
cytes which are localised in the subepithelial region
Abbreviations: ELISA, enzyme linked immunosorbent assays;
IEC, intestinal epithelial cells; IL-1β, interleukin 1 beta; LPL, lam-
ina propria lymphocytes; MLR, mixed lymphocyte reactions;
PBL, peripheral blood lymphocytes; PBMC, peripheral blood
mononuclear cells; RT-PCR, reverse transcription-polymerase
chain reaction; TNF-α, tumour necrosis factor alpha.
*Address correspondence to Dr. Stephanie Blum, Department
of Immunology, Nestlé Research Centre, Vers-chez-les-Blanc, Ch-
1000 Lausanne 26, Switzerland. Fax: 0041―21―785―8925.
195

196
D. HALLER ET AL
also contribute to the suppression of lymphocyte activa-
tion suggesting that accessory cell function may be an
additional target for the immunomodulatory effect of
the mucosal environment (36, 44).
obtain final cell densities of 1×106 and 1×107 colony
forming units (cfu/ml) in RPMI 1640 medium (Gibco
BRL).
Cell culture. Human intestinal epithelial cell (IEC)
Intestinal epithelial cells which are the first cells in the
frontline interacting with intestinal bacteria, are con-
sidered to participate in the initiation and regulation of
mucosal immune response (5, 26, 34, 39). Experiments
in germ-free animals have shown that lymphoid popu-
lations in the lamina propria are considerably reduced in
the germ-free intestine, but assume its normal appearance
of physiological inflammation after bacterial colonisation
(4, 9, 28). We have recently shown that commensal
non-pathogenic bacteria of different origin have the abil-
ity to elicit a characteristic cytokine response in leukocyte
sensitised CaCO-2 cells in vitro and deliver a discrimi-
native signal to underlying immunocompetent cells (25).
We provided evidence that bi-directional cross talk
between IEC and immunocompetent cells constitutes a
crucial step in the response to non-pathogenic bacteria at
the mucosal surface.
lines CaCO-2 and HT-29 (passage 40
―
60) were seeded
(2×105 cells/well) on 10.5 mm cell culture inserts (0.4
µm nucleopore size; Becton Dickinson) and were placed
in 12-well tissue culture plates (Nunc). CaCO-2 cells
were cultured for 18
―
21 days at 37 C/10% CO2 in
DMEM (glutamine, high glucose; Amimed) supple-
mented with 20% decomplemented fetal calf serum (56
C, 30 min) (FCS, Amimed), 1% MEM non-essential
amino acids (Gibco BRL) and 0.1% penicillin/strepto-
mycin (10,000 IU/ml/10,000 UG/ml, Gibco BRL). HT-
29 cells were cultured for 12
―
15 days at 37 C/5% CO2 in
DMEM (glutamine, high glucose, Amimed) and 0.1%
penicillin/streptomycin (10,000 IU/ml/10,000 UG/ml,
Gibco BRL). Transepithelial electrical resistance (TEER)
(ohm/cm2) was determined continuously in confluent
CaCO-2 and HT-29 monolayers using a MultiCell-ERS
electrode (voltmeter/ohmmeter).
The aim of this study was to investigate the role of
lymphoid and myeloid cell populations in the regulation
of immune-mediated activation of IEC after the challenge
with non-pathogenic food derived and intestinal bacteria.
We provide evidence that IEC conditioned monocytes
acquire a phenotype similar to lamina propria mono-
cytes, which in turn, control inflammatory cytokine
expression in IEC and modulate lymphocyte activation in
vitro. The role of bacterial activated IEC in the devel-
opment of immunosuppressive monocytes will be dis-
cussed in the context of pathological conditions, such as
inflammatory bowel disease (IBD).
Purification of leukocyte subpopulations from human
peripheral blood mononuclear cells. Human peripheral
blood mononuclear cells (PBMC) from healthy volun-
teers were purified from buffy coats (Blood Transfu-
sion Center, Lausanne) using Ficoll-Hypaque 1077
(Pharmacia) gradient centrifugation (500×g, 30 min).
PBMC were incubated for 2 hr at 37 C and 5% CO2 on
225-cm2 tissue culture plates (Costar) to allow adherence.
Nonadherent peripheral blood lymphocytes (PBL) were
separated from adherent cells by aspiration. PBL were
incubated with CD4, CD8, CD19 magnetic MicroBeads
(Miltenyi Biotech) and separated by magnetic cell sort-
ing (MACS) using positive selection technique. Mono-
cytes were enriched from PBMC by depletion of T cells,
B cells, NK cells, dendritic cells and basophils using indi-
rect labelling with hapten-conjugated CD3, CD7, CD19,
CD45RA, CD56, anti-IgE monoclonal antibodies (mAb)
and MACS MicroBeads coupled to an antihapten mon-
oclonal antibody (Monocyte Isolation Kit, Miltenyi
Biotech). Finally, cells were diluted in RPMI 1640 con-
taining 10% FCS (56 C, 30 min) and gentamicin (100
µg/ml, Gibco BRL) to a final density of 2×106 cells/ml.
Where indicated lower cell numbers were used (5×105
to 1×106 cells/ml).
Materials and Methods
Bacteria and growth condition. The non-pathogenic
Escherichia coli D2241 of human faecal origin was
obtained from the Statens Serum Institute (International
Escherichia and Klebsiella Center, Copenhagen, Den-
mark) and was grown in brain-heart-infusion broth (BHI)
at 37 C. Intestinal lactobacilli, Lactobacillus johnsonii
La1 (Strain collection, Nestlé Research Centre, Lau-
sanne, Switzerland) and Lactobacillus gasseri (ATCC
33323) were grown in MRS broth (14) without acetate at
37 C. Lactobacillus sakei LTH 681 (Strain Collection,
Hohenheim University; Institute of Food Technology,
Stuttgart, Germany), isolated from fermented food, was
grown in the same broth at 30 C. All bacteria were har-
vested by centrifugation (3,000×g, 10 min) after 24 hr
of cultivation at stationary growth phase. Bacteria were
washed three times with phosphate buffered saline (PBS)
(1×, pH 7.2, Gibco BRL) and subsequently diluted to
Flow cytometry. Cell staining was performed for 20
min at 4 C with saturating concentrations of the fol-
lowing mouse anti-human mAbs: FITC-conjugated anti
CD4- (SK3) (IgG1, Becton Dickinson), CD8- (SK1)
(IgG1, Becton Dickinson), CD19- (4G7) (IgG1, Becton
Dickinson), CD58- (MY31) (IgG1, Becton Dickinson),
CD33- (HIM3-4) (IgG1, Pharmingen), CD16- (3G8)
(IgG1, Pharmingen), HLA-DR- (L243) (IgG2a, Becton

IMMUNOREGULATORY CD14low MONOCYTES IN IEC CO-CULTURES
197
Dickinson), CD80- (L307.4) (IgG1, Pharmingen), CD86-
(2331) (IgG1, Pharmingen) mAb; biotin-conjugated anti
CD11b- (ICRF44) (IgG1, Pharmingen) mAb followed by
Avidin-FITC (1:100, Pharmingen) and PE-conjugated
anti-CD14 mAb (MøP9) (IgG2b, Becton Dickinson).
Isotype controls: FITC-conjugated mouse IgM, IgG1,
IgG2a mAb (G155-228, MOPC-21, G155-178, Pharmin-
gen) and PE-conjugated mouse mAb (27-35, IgG2b,
Pharmingen). The samples were analysed after double
staining using a FACScan® (Becton Dickinson).
Cell viability. Cell viability was routinely tested by
trypan exclusion. Discrimination between viable, apo-
ptotic and necrotic cells was performed by flow cytom-
etry using the VybrantTM Apoptosis Assay Kit (Molecu-
lar Probes).
Intestinal epithelial cell/leukocyte co-cultures. Trans-
well cultures with confluent CaCO-2 or HT-29 mono-
layers were established as previously described (23).
Freshly purified human PBMC, lymphocyte subpopula-
tions or CD14high monocytes were added to the basolateral
compartment at cell densities of 2×106/ml. IEC/leuko-
cyte co-cultures were stimulated with bacteria by adding
1×106 or 1×107 cfu/ml non-pathogenic E. coli or lac-
tobacilli to the apical surface of IEC monolayers and
incubated for 4, 16, 36 or 72 hr at 5% CO2 and 37 C. To
prevent bacterial growth Gentamicin (150 µg/ml) was
added to the apical compartment of the cultures after 4
hr of incubation. Where indicated, neutralising anti-
IL-10 mAb (10 µg/well, 500-M86, BioConcept) and
recombinant human (rh) IL-10 (10 pg/well, R&D Sys-
tems) were added to the basolateral compartment of the
co-cultures.
thiocyanate/phenol/chloroform method (Micro RNA
Isolation Kit, Stratagene). Total RNA (0.5 µg), 1 mM of
each dNTP, 2.5 U/ml MuLV reverse transcriptase (Perkin
Elmer), and specific 3' primers coding for the human
cytokines TNF-α, IL-8 and β-actin were incubated at 42
C for 30 min. PCR amplification and subjection of
PCR products to gel electrophoresis was performed as
previously described (23).
ELISA. Quantification of IL-10, and IL-1Ra (R&D
Systems) in the supernatants of IEC/leukocyte co-cultures
was performed by ELISA.
Mixed lymphocyte reaction. Mixed lymphocyte reac-
tion (MLR) was performed with freshly purified allo-
geneic monocyte depleted peripheral blood lympho-
cytes (PBL) and in vitro conditioned monocytes at vary-
ing concentrations (1×105, 5×104, 1×104, 5×103, 1×
103 cells/well). Monocytes were harvested from transwell
cultures after 3 days of culture on plastic (Mo 1), co-cul-
ture with HT-29 cells (Mo 2) and co-culture with L.
sakei (107 cfu/ml) activated HT-29 cells (Mo 3). Finally,
serial dilutions of the three differently matured monocytes
from the same donor were incubated with a fixed number
of allogeneic lymphocytes (2×105 cells/well). MLR
cultures were maintained for 5 days at 5% CO2 and 37 C.
During the last 18 hr of cultures 1 µCi of [3H]-thymidine
(Amersham) was added to each well. Thereafter, cells
were harvested on filter mats and [3H]-thymidine incor-
poration was determined by liquid scintillation counting
(TopCount, Packard).
Statistical analysis. Values are given as mean (SD) of
triplicate measurements. All results were confirmed for
at least three different donors in independent experi-
ments. Significance was tested by Mann-Whitney U
test (Statistica, Statsoft).
RNA extraction and amplification by RT-PCR. Total
RNA from IEC was isolated using the acid guanidinium
Fig. 1. Differential activation of CaCO-2 and HT-29 cells by non-pathogenic bacteria. Reverse transcription-polymerase chain reaction
(RT-PCR) was used to determine tumour necrosis factor alpha (TNF-α) mRNA expression in CaCO-2 and HT-29 cells on stimulation
of intestinal epithelial cells (IEC)/leukocyte co-cultures or IEC alone with non-pathogenic E. coli, L. sakei, L. gasseri and L. johnsonii
(16 hr, 106 and 107 cfu/ml), respectively. Culture medium alone was used as a control. Results represent one of two independent exper-
iments.

198
D. HALLER ET AL
Fig. 2. Peripheral blood lymphocytes drive activation of HT-29 cells after bacterial stimulation. Reverse transcription-polymerase chain
reaction (RT-PCR) analysis was used to determine tumour necrosis factor alpha (TNF-α) and interleukin (IL)-8 mRNA expression in
HT-29 and CaCO-2 cells after stimulation with L. sakei and E. coli (16 hr, 1×107 cfu/ml), respectively. (A) HT-29 and (B) CaCO-2 cells
＋
＋
＋
＋
were reconstituted with CD4 and CD8 T cells, CD19 B cells, CD14 monocytes and peripheral blood mononuclear cells (PBMC)
(2×106 cells/ml). The absence of immunocompetent cells (no cells) was used as a control. Results represent one of two independent exper-
iments.
Fig. 3. Differential expression of TNF-α and IL-10 mRNA in leukocyte populations of CaCO-2 transwell cultures. CaCO-2 cell trans-
well cultures were apically stimulated with E. coli (16 hr, 1×107 cfu/ml). Reverse transcription-polymerase chain reaction (RT-
PCR) analysis was used to determine tumour necrosis factor alpha (TNF-α) (A) and interleukin (IL)-10 (B) in underlying PBMC, PBL,
＋
＋
＋
＋
T cells (CD4 :CD8 ratio 1:1), B cells (CD19 ), monocytes (CD14 ), as well as combinations of T cells and monocytes
＋
＋
＋
＋
＋
＋
(CD4 :CD8 :CD14 ratio 1:1:1) or T/B cells (CD4 :CD8 :CD19 ratio 1:1:1) (2×106 total cells/ml). The absence of immunocompetent
cells (no cells) was used as a control. Results represent one of two independent experiments.
induced TNF mRNA and IL-8 mRNA expression in
Results
IEC after 12 hr of incubation. The strong activation of
IEC determined between 14
―
20 hr of incubation was
Peripheral Blood Lymphocytes Drive Activation of IEC
after Challenge with Non-Pathogenic Bacteria
Previous results (25) demonstrated that the stimulation
of CaCO-2/PBMC co-cultures with E. coli and L. sakei
completely down-regulated after 36 hr. In this study, the
stimulation of CaCO-2 and HT-29 transwell cultures
by non-pathogenic E. coli, L. sakei, L. gasseri and L.
johnsonii for 16 hr resulted in a characteristic activation

IMMUNOREGULATORY CD14low MONOCYTES IN IEC CO-CULTURES
199
pattern of IEC, when PBMC (2×106/well) were present
in the basolateral compartment. As shown in Fig. 1,
TNF-α mRNA expression in IEC was strongly induced
by 107 cfu/ml Gram negative E. coli and Gram positive L.
sakei, while the inflammatory cytokine response in HT-
29 and CaCO-2 cells induced by Gram positive intestinal
lactobacilli such as L. johnsonii or L. gasseri remained at
low levels. E. coli induced low levels of TNF-α mRNA
expression in HT-29 cells but not in CaCO-2 cells, even
in the absence of immunocompetent cells. These results
are in accordance with our recently published data (25).
To identify the leukocyte population involved in IEC
with B cells and monocytes. On the other hand, RT-PCR
analysis of the same samples showed IL-10 mRNA
expression in PBMC, monocytes as well as a combina-
tion of T cells and monocytes (CD4:CD8:CD14 ratio
1:1:1), but not in T cells (CD4:CD8 ratio 1:1), B cells and
the combination of T/B cells (CD4:CD8:CD19 ratio
1:1:1) (Fig. 3B).
Previous results showed that the Gram negative E.
coli but not Gram positive L. sakei induced IL-10 expres-
sion in PBMC (24). To further elucidate whether mono-
cytes exert immunoregulatory functions in IEC co-cul-
tures, we measured the secretion of regulatory mediators
such as IL-10 and IL-1 receptor antagonist (IL-1Ra) in
the basolateral compartment of IEC/monocyte co-cultures
after the stimulation with L. sakei. HT-29 cells were
stimulated with L. sakei (107 cfu/ml) for 4, 16 and 36 hr.
＋
＋
activation, MACS purified CD4 and CD8 T cells,
＋
CD19 B cells or CD14high monocytes (2×106/well)
were used to reconstitute HT-29 and CaCO-2 transwell
cultures. As shown in Fig. 2A, HT-29 stimulation with
L. sakei (107 cfu/ml) for 16 hr resulted in the expression
of TNF-α mRNA in IEC, when either total PBMC,
＋
＋
＋
CD4 , CD8 T cells or CD19 B cells were present in
the basolateral compartment. Induction of IL-8 mRNA
＋
＋
was mediated by PBMC, CD4 and CD8 T cells, but
＋
not by CD19 B cells after bacterial stimulation.
Notably, purified CD14high monocytes failed to mediate
activation of HT-29 cells after bacterial stimulation.
Figure 2B shows E. coli-induced TNF-α mRNA expres-
sion in CaCO-2 cells in the presence of total PBMC,
＋
＋
CD4 and CD8 T cells. Low TNF-α mRNA expres-
sion was induced by CD14high monocytes, whereas
＋
CD19 B cells failed to mediate epithelial cell activation.
Induction of IL-8 mRNA was only induced, when either
＋
total PBMC or CD4 T cells were present in CaCO-2 co-
＋
cultures. In summary, total PBMC and CD4 T cells
consistently induced TNF-α and IL-8 mRNA expression
in both IEC lines, whereas CD14low monocytes and
＋
CD19 B cells revealed a low potential to trigger an
inflammatory cytokine response in bacteria stimulated
IEC. Unstimulated IEC/leukocyte co-cultures were not
activated suggesting that alloantigens released by IEC are
not responsible for the activation of IEC (data not
shown).
Constitute an Immunoregulatory Subpopulation in Bac-
teria Challenged IEC/Leukocyte Co-Cultures
To further dissect the function of different leukocyte
subpopulations in IEC co-cultures, we stimulated IEC
with bacteria and investigated TNF-α and IL-10 mRNA
expression in underlying immune cells. First, CaCO-2
cells were stimulated for 16 hr with E. coli (107 cfu/ml)
and RT-PCR analysis of IEC co-cultured leukocyte pop-
ulations was performed. Figure 3A shows low expres-
sion of TNF-α mRNA in monocytes, a slightly higher
expression in B cells, and strong expression in total
PBMC, PBL, T cells as well as T cell combinations
Fig. 4. IL-10 and IL-1Ra secretion in HT-29/monocyte co-cultures
after stimulation with non-pathogenic bacteria. HT-29/mono-
cyte co-cultures were stimulated with L. sakei (1×107 cfu/ml)
(black bar) or culture medium (no treatment) (gray bar). Secretion
of IL-10 and IL-1 receptor antagonist (Ra) was determined
(pg/ml) in the basolateral compartment of transwell cultures
after 4, 16 and 36 hr. Data are mean (SD) of triplicate values and
represent one of three independent experiments.

200
D. HALLER ET AL
Fig. 5. Monocyte-derived IL-10 down-regulates bacterial induced activation of IEC. Reverse transcription-polymerase chain reaction (RT-
PCR) analysis was used to determine tumour necrosis factor alpha (TNF-α) and interleukin (IL)-8 mRNA expression in CaCO-2 and
HT-29 cells after bacterial stimulation. (A) CaCO-2 cells were co-cultured with monocyte-depleted peripheral blood lymphocytes (PBL,
left panel) and peripheral blood mononuclear cells (PBMC, right panel) and stimulated with E. coli (36 hr, 1×107 cfu/ml). (B) HT-29
cells were co-cultured with PBMC and stimulated with L. sakei (36 hr, 1×107 cfu/ml) or E. coli (36 hr, 1×107 cfu/ml). Where indicated
(A) recombinant human (rh) IL-10 (10 pg/ml) or (B) neutralising anti-IL-10 monoclonal antibodies (mAb) (10 µg/ml) were added to the
basolateral compartment. Controls were performed in the absence of anti-IL-10 mAb or rh IL-10. Isotype control antibodies did not change
the results. Results represent one of three independent experiments.
As shown in Fig. 4, IL-1Ra secretion was already
induced in HT-29/monocyte cultures after 4 hr of bacte-
rial stimulation, however maximal secretion of IL-1Ra
(13,850 pg/ml) and IL-10 (40,325 pg/ml) was detectable
after 16 hr. The concentrations of IL-1Ra (10,825 pg/ml)
and IL-10 (31,200 pg/ml) remained at relatively high lev-
els after 36 hr incubation. These results demonstrate that
bacterial activated CaCO-2 and HT-29 cells induce the
expression of immunoregulatory mediators in co-cul-
tured monocytes. L. sakei was used in this study to
ensure that not only translocated bacterial products medi-
ate IL-10 induction in IEC/monocyte co-cultures.
co-cultures for 36 hr with E. coli. As shown in Fig. 5A
(left panel), TNF-α mRNA expression in bacteria stim-
ulated IEC was sustained in the absence of monocytes
(CaCO-2/PBL cultures). The addition of rh IL-10 (10
pg/ml) to CaCO-2/PBL co-cultures inhibited the expres-
sion of TNF-α mRNA in CaCO-2 cells, demonstrating
the pivotal role of IL-10 in the regulation of epithelial cell
activation. In the same experiment (Fig. 5A, right panel),
we demonstrated that the presence of neutralising anti-IL-
10 mAbs (10 µg/ml) in E. coli stimulated CaCO-2/
PBMC co-cultures sustained the expression of TNF-α
mRNA at late time points (36 hr). Similarly, when neu-
tralising anti-IL-10 mAbs (10 µg/ml) were added to the
basolateral compartment of L. sakei or E. coli (107
cfu/ml) stimulated HT-29/PBMC co-cultures, TNF-α
and IL-8 mRNA expression in IEC was sustained at 36
hr. These results may demonstrate that monocyte-derived
IL-10 plays an important regulatory role in bacteria
stimulated IEC/leukocyte cultures. Antibodies alone
did not induce IEC activation. Specificity for the effects
of anti-IL-10 mAb was controlled using Ig isotype con-
trol antibodies.
Monocyte-Derived IL-10 Down-Regulates Proinflam-
matory Cytokine Expression in Bacteria-Activated IEC
after 36 hr Stimulation
We showed that monocytes have low potential to
induce a bacteria-dependent activation of proinflamma-
tory cytokine expression in IEC and at the same time,
produce immunoregulatory cytokines such as IL-10.
Our previous study showed that the expression TNF-α
mRNA in E. coli stimulated CaCO-2 cells was first
induced after 12
―
20 hr and down-regulated after 36 hr
(25). To demonstrate that monocyte-derived IL-10
down-regulates a bacteria induced epithelial cell activa-
tion, we depleted PBMC from monocytes (peripheral
blood lymphocytes, PBL) and stimulated CaCO-2/PBL
IEC and Bacterial Stimulated IEC Modulate Cell Surface
Antigen Expression of Monocytes
To investigate the influence of IEC on the phenotype
of co-cultured monocytes, the expression cell surface

IMMUNOREGULATORY CD14low MONOCYTES IN IEC CO-CULTURES
201
(A)
(B)
Fig. 6. Intestinal epithelial cells induce phenotype changes in peripheral blood monocytes. Peripheral blood CD14high monocytes
were co-cultured with HT-29 cells for 36 hr (A) and 72 hr (B) in the presence of culture medium (Mo 2) or L. sakei (1×107 cfu/ml) (Mo
3). Control monocytes were cultured in 12-well plates in the absence of HT-29 cells (Mo 1). FACS analysis was used to determine cell
surface expression of CD14, CD16 and CD11b (% positive cells). Isotype controls (IgG2b, IgG1, IgG2a) were performed. Results rep-
resent one of three independent experiments.
markers on monocytes were analysed after their culture
in the absence of IEC (Mo 1), their culture in the pres-
ence of IEC (Mo 2), and their culture in the presence of
bacterial stimulated IEC (Mo 3). Figure 6A shows the
change of CD14high monocytes to a CD14low CD16low phe-
notype in IEC co-cultures at 36 hr. In the absence of IEC
(Mo 1) the predominant monocyte phenotype was
CD14high CD16high, whereas in the presence of HT-29
cells (Mo 2) the CD14/CD16 double positive population
was markedly reduced (60% to 28%). This phenotype

202
D. HALLER ET AL
Fig. 7. Capability of monocytes to stimulate allogeneic lymphocyte response. Peripheral blood CD14 positive
monocytes were co-cultured with HT-29 cells for 3 days in the presence of culture medium (triangle down) or
L. sakei (1×107 cfu/ml) (triangle up). Control monocytes were cultured in 12-well plates in the absence of HT-
29 cells (square). Thereafter, mixed lymphocyte reaction (MLR) was performed with allogeneic peripheral blood
lymphocytes (PBL) (2×105 cells/well) and monocytes at varying concentrations (1×105, 5×104, 1×104, 5×
103, 1×103 cells/well). MLR was maintained for 5 days and [3H]-thymidine incorporation was determined (cpm).
Data are mean (SD) of triplicate values and represent one of three independent experiments.
changes were even stronger pronounced when HT-29
cells were stimulated with L. sakei (60% to 5%) (Mo 3).
Although the distinct CD14high monocyte population
changed to a more CD14low phenotype, the total per-
centage of CD14 positive cells remained at relatively high
numbers (55% for Mo 2 and 37% for Mo 3).
Decreased Capacity of IEC Co-Cultured Monocytes to
Stimulate Allogeneic Lymphocyte Responses
One important function of antigen presenting cells
(APC) is the stimulation of lymphocytes. Mixed-lym-
phocyte reactions (MLR) can be used to assess to poten-
tial of APC to stimulate proliferation of allogeneic lym-
phocytes (38). When the different in vitro matured
Based on these results, we performed a more detailed
analysis of the monocyte phenotype after 3 days of cul-
ture. As shown in Fig. 6B (Mo 2), co-cultivation of
CD14high monocytes with HT-29 cells resulted in the
down-regulation of CD14 (55% to 35% positive cells)
and CD16 (43% to 20%), while CD11b remained at
high levels (78% to 87%). The expression of co-stimu-
latory molecules such as CD80 (63% to 51%), CD86
(66% to 51%) and CD58 (49% to 31%) were also
reduced (data not shown). The stimulation of HT-29 cells
with L. sakei (Fig. 6A, Mo 3) resulted in a further down-
regulation of CD14 (35% to 19%) and CD16 (20% to
8%), whereas the expression of CD11b (87% to 84%)
and co-stimulatory molecules remained unchanged
(CD80: 51% to 48%; CD86: 51% to 50%; CD58: 31% to
30%) (data not shown). IEC and bacterial stimulated IEC
did neither influence the expression of CD33 (lineage
marker) nor HLA-DR (data not shown). Thus, IEC co-
cultured CD14high peripheral blood monocytes acquired a
CD14low CD16low CD11bhigh CD33high HLA-DRhigh CD80low
CD86low CD58low. Cell viability after 3 days was ＞95%.
The number of apoptotic or necrotic cells did not exceed
5% (data not shown).
monocytes (Mo 1
―
3) were investigated for their capaci-
ty to stimulate proliferation of alloreactive human PBL,
only the plastic adherent monocytes (Mo 1) were able to
stimulate significant proliferation (stimulator/responder
cell ratio 1:4, 1:2). In contrast, those monocytes, matured
in the presence of HT-29 or L. sakei activated HT-29
cells, could not trigger a substantial lymphocyte response
(Fig. 7). Similar results were obtained when purified
＋
CD4 T cells were used as responder cells (data not
shown).
Discussion
Intestinal monocytes, which constitute 10
―
20% of
mononuclear cells in the lamina propria, differ marked-
ly in phenotype and function from peripheral blood
monocytes (16, 40). The classical monocyte-specific
surface antigens CD14 (LPS co-receptor), CD11b (com-
plement receptor 3, CR3) and CD16 (FcγIII receptor) are
expressed at low levels in the normal intestinal mucosa.
This is thought as one mechanism to prevent activation
induced by trace amounts of translocated bacterial prod-
ucts (33, 46). Under pathological conditions, such as

IMMUNOREGULATORY CD14low MONOCYTES IN IEC CO-CULTURES
203
active inflammatory bowel diseases (IBD), an increased
fraction of monocytes with a monocyte-like phenotype
appears in the lamina propria (1, 22, 23, 48, 49).
Rugtveit et al. (50) showed that these CD14high monocytes
in IBD mucosa represent a newly recruited subset of
intestinal monocytes extravasated from the peripheral
blood, harbouring an increased potential for the pro-
duction of proinflammatory cytokines compared to the
resident CD14low population, and therefore, may be
involved the development of IBD (8).
that L. sakei induced the secretion of IL-12 and IFN-γ but
not IL-10 in PBMC cultures (24), demonstrating the
capability of IEC to modulate immune responses. It
was remarkable, that at later time points monocyte-
derived IL-10 antagonised the lymphocyte-dependent
activation of IEC with respect to TNF-α, and to a lesser
extent IL-8 gene expression. The anti-inflammatory
cytokine IL-10 has been reported to play an essential role
in the control of normal gut homeostasis (2, 51). In
active inflammatory bowel disease, associated with
increased proinflammatory cytokines (42, 54), IL-10
secretion has been shown to down-regulate proinflam-
matory cytokine response in IBD derived lamina propria
mononuclear cells and peripheral blood monocytes (52).
In our model system, the IL-10 response was exclusive-
ly detected in monocytes and therefore might have inhib-
ited TNF-α expression by an autocrine/paracrine, nega-
tive feed back regulation (35). Kucharzik et al. (29)
showed that CaCO-2 cells prime lamina propria mononu-
clear cells to induce IL-10 secretion. The authors pro-
posed that monocytes may be selectively targeted by
epithelial cell derived mediators such as TGF-β1 or
prostaglandin-E2 (PGE), resulting in the secretion of
IL-10.
The modulation of lamina propria lymphocyte acti-
vation by local monocytes was reported to be partially
based on their low expression of the co-stimulatory mol-
ecules CD80 (B7.1), CD86 (B7.2) and CD58 for T cell
accessory receptors CD28/CTLA-4 and CD2 (44). The
appearance of CD14high CD80high monocytes in IBD
lesions was reported to change the normal anergic
mucosal state towards an enhanced Th1-type of immune
activation (47, 53), leading to a loss of tolerance against
the indigenous bacterial flora. We demonstrated that
under the influence of IEC, the percentage expression of
co-stimulatory molecules, were down regulated, while
expression of MHC class II molecules was conserved. In
fact, IEC conditioned monocytes had reduced potential to
trigger an allogeneic lymphocyte response. CD14high
peripheral blood monocytes, which are recruited to the
intestinal mucosa, undergo differentiation near the epithe-
lial surfaces. Thus, the influence of the intestinal epithe-
lium on this particular monocyte population might be of
critical importance in the modulation of the local T cell
responses and induction of oral tolerance to luminal
antigens (37). In vitro, this regulation is bidirectional,
since the co-cultured PBMC inhibited bacterial induced
TNF-α and IL-8 mRNA expression, a process mediated
in part by monocyte-derived IL-10. Notably, commen-
sal bacteria further promoted the induction of CD14low
CD16low immunoregulatory monocytes by IEC, suggest-
ing that bacterial cross talk at the mucosal surface could
be beneficial for the host (32).
In this in vitro study, we demonstrated that under the
influence of intestinal epithelial cells (IEC), peripheral
blood CD14high monocytes changed to a CD14low phe-
notype with immunoregulatory functions. A recent
study from Spöttl et al. (56) showed for the first time that
IEC cultured in multicellular spheroids induce the dif-
ferentiation of CD14high peripheral blood monocytes in
intestine-like macrophage phenotype. In addition, we
showed that the development of a CD14low monocyte
phenotype is further pronounced in the presence of bac-
teria activated IEC, suggesting that non-pathogenic bac-
teria/epithelial cell cross talk may help to maintain local
immune homeostasis. The absence of increased numbers
of apoptotic or necrotic cells during co-culture indicated
that the loss of CD14high expressing cells may be a con-
sequence of monocyte differentiation and/or receptor
shedding rather than cell death (3). This was supported
by the observation that the panmyolilic differentiation
marker CD33, reliable for the identification of intestinal
tissue monocytes (46), and CD11b remained high. The
development of CD14high CD16high tissue monocytes,
observed under certain inflammatory conditions (6, 7, 17,
21, 30) was significantly suppressed under the influ-
ence of IEC and bacteria stimulated IEC, even though the
kinetic in the development of a CD14low and CD16low
phenotype differed for the two cell surface markers.
Although soluble bacterial products such as lipopolysac-
charide (LPS), peptidoglycan-polysaccharide (PG-PS), or
formylated chemotactic oligopeptides (fMLP) (10, 55,
57), which have been shown to initiate inflammatory
processes, may translocate to some extent through the
epithelial cell monolayer, the stimulation of IEC with
non-pathogenic E. coli or L. sakei facilitated the devel-
opment of CD14low CD16low monocytes. The role of pat-
tern recognition receptors such TLR4 and TLR2 in the
initiation of non-pathogenic bacteria-induced IEC acti-
vation is under current investigation.
＋
Lymphoid populations preferentially CD4 T cells
were shown to drive the activation of IEC by intestinal E.
coli or food derived L. sakei, through a yet undefined
mechanism, while monocytes were identified as a
immunoregulatory cell population, secreting high
amounts of IL-1Ra and IL-10. A previous study showed

204
D. HALLER ET AL
um for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:
130 135.
References
―
15) D’Haens, G.R., Geboes, K., Peeters, M., Baert, F., Pen-
ninckx, F., and Rutgeerts, P. 1998. Early lesions of recurrent
Crohn’s disease caused by infusion of intestinal contents
1) Allison, M.C., and Poulter, L.W. 1991. Changes in pheno-
typically distinct mucosal macrophage populations may be a
prerequisite for the development of inflammatory bowel
in excluded ileum. Gastroenterology 114: 262―267.
disease. Clin. Exp. Immunol. 85: 504
―
509.
16) Donnellan, W.L. 1965. The structure of the colonic mucosa.
The epithelium and subepithelial reticulohistiocytic com-
2) Autschbach, F., Braunstein, J., Helmke, B., Zuna, I., Schur-
mann, G., Niemir, Z.I., Wallich, R., Otto, H.F., and Meuer,
S.C. 1998. In situ expression of interleukin-10 in nonin-
flamed human gut and in inflammatory bowel disease. Am.
J. Pathol. 153: 121―130.
3) Bazil, V., and Strominger, J.L. 1991. Shedding as a mecha-
nism of down-modulation of CD14 on stimulated human
plex. Gastroenterology 49: 496―514.
17) Draude, G., von Hundelshausen, P., Frankenberger, M.,
Ziegler-Heitbrock, H.W., and Weber, C. 1999. Distinct scav-
enger receptor expression and function in the human
CD14(＋)/CD16(＋) monocyte subset. Am. J. Physiol. 276:
H1144―H1149.
monocytes. J. Immunol. 147: 1567
4) Berg, R.D., and Savage, D.C. 1972. Immunological respons-
es and microorganisms indigenous to the gastrointestinal
―
1574.
18) Duchmann, R., Kaiser, I., Hermann, E., Mayet, W., Ewe, K.,
and Meyer, Z.B.K. 1995. Tolerance exists towards resident
intestinal flora but is broken in active inflammatory bowel dis-
tract. Am. J. Clin. Nutr. 25: 1364
―1371.
ease (IBD). Clin. Exp. Immunol. 102: 448―455.
5) Blumberg, R.S., Terhorst, C., Bleicher, P., McDermott, F.V.,
Allan, C.H., Landau, S.B., Trier, J.S., and Balk, S.P. 1991.
Expression of a nonpolymorphic MHC class I-like molecule,
CD1D, by human intestinal epithelial cells. J. Immunol.
19) Duchmann, R., May, E., Heike, M., Knolle, P., Neurath,
M., and Meyer, Z.B.K. 1999. T cell specificity and cross reac-
tivity towards enterobacteria, bacteroides, bifidobacterium,
and antigens from resident intestinal flora in humans. Gut 44:
147: 2518
―2524.
812―818.
6) Blumenstein, M., Boekstegers, P., Fraunberger, P., Andreesen,
R., Ziegler-Heitbrock, H.W., and Fingerle-Rowson, G. 1997.
20) Duchmann, R., Schmitt, E., Knolle, P., Meyer, Z.B.K., and
Neurath, M. 1996. Tolerance towards resident intestinal
flora in mice is abrogated in experimental colitis and restored
by treatment with interleukin-10 or antibodies to interleukin-
＋
Cytokine production precedes the expansion of CD14
＋
CD16 monocytes in human sepsis: a case report of a patient
with self-induced septicemia. Shock 8: 73
―75.
12. Eur. J. Immunol. 26: 934―938.
7) Brauner, A., Lu, Y., Hallden, G., Hylander, B., and Lundahl,
J. 1998. Difference in the blood monocyte phenotype
between uremic patients and healthy controls: its relation to
monocyte differentiation into macrophages in the peritoneal
21) Fingerle, G., Pforte, A., Passlick, B., Blumenstein, M.,
Strobel, M., and Ziegler-Heitbrock, H.W. 1993. The novel
＋
＋
subset of CD14 /CD16 blood monocytes is expanded in
sepsis patients. Blood 82: 3170 3176.
―
cavity. Inflammation 22: 55
―
66.
22) Grimm, M.C., Pavli, P., Van de Pol, E., and Doe, W.F. 1995.
＋
8) Casini-Raggi, V., Kam, L., Chong, Y.J., Fiocchi, C., Pizarro,
T.T., and Cominelli, F. 1995. Mucosal imbalance of IL-1 and
IL-1 receptor antagonist in inflammatory bowel disease. A
novel mechanism of chronic intestinal inflammation. J.
Evidence for a CD14 population of monocytes in inflam-
matory bowel disease mucosa—implications for pathogen-
esis. Clin. Exp. Immunol. 100: 291―297.
23) Grimm, M.C., Pullman, W.E., Bennett, G.M., Sullivan, P.J.,
Pavli, P., and Doe, W.F. 1995. Direct evidence of mono-
cyte recruitment to inflammatory bowel disease mucosa. J.
Immunol. 154: 2434
―2440.
9) Cebra, J.J. 1999. Influences of microbiota on intestinal
immune system development. Am. J. Clin. Nutr. 69: 1046S
―
Gastroenterol. Hepatol. 10: 387―395.
1051S.
24) Haller, D., Blum, S., Bode, C., Hammes, W.P., and Schiffrin,
E.J. 2000. Activation of human peripheral blood mononuclear
cells by nonpathogenic bacteria in vitro: evidence for NK
10) Chester, J.F., Ross, J.S., Matt, R.A., and Weitzmann, S.A.
1985. Acute colitis produced by chemotactic peptides in
rats and mice. Am. J. Pathol. 121: 284
―290.
cells as primary targets. Infect. Immun. 68: 752―759.
11) Christ, A.D., Colgan, S.P., Balk, S.P., and Blumberg, R.S.
1997. Human intestinal epithelial cell lines produce fac-
tor(s) that inhibit CD3-mediated T-lymphocyte prolifera-
25) Haller, D., Bode, C., Hammes, W.P., Pfeifer, A.M., Schiffrin,
E.J., and Blum, S. 2000. Non-pathogenic bacteria elicit a dif-
ferential cytokine response by intestinal epithelial cell/leu-
tion. Immunol. Lett. 58: 159
―165.
cocyte co-cultures. Gut 47: 79―87.
12) Cong, Y., Brandwein, S.L., McCabe, R.P., Lazenby, A.,
Birkenmeier, E.H., Sundberg, J.P., and Elson, C.O. 1998.
26) Jung, H.C., Eckmann, L., Yang, S.K., Panja, A., Fierer, J.,
Morzycka-Wroblewska, E., and Kagnoff, M.F. 1995. A dis-
tinct array of proinflammatory cytokines is expressed in
human colon epithelial cells in response to bacterial invasion.
＋
CD4 T cells reactive to enteric bacterial antigens in spon-
taneously colitic C3H/HeJBir mice: increased T helper cell
type 1 response and ability to transfer disease. J. Exp. Med.
J. Clin. Invest. 95: 55
27) Kagnoff, M., and Eckmann, L. 1997. Epithelial cells as sen-
sors for microbial infection. J. Clin. Invest. 100: 6 10.
―65.
187: 855―864.
13) Darfeuille-Michaud, A., Neut, C., Barnich, N., Lederman, E.,
Di Martino, P., Desreumaux, P., Gambiez, L., Joly, B., Cor-
tot, A., and Colombel, J.F. 1998. Presence of adherent
Escherichia coli strains in ileal mucosa of patients with
―
28) Kenworthy, R. 1971. Observations on the reaction of the
intestinal mucosa to bacterial challenge. J. Clin. Pathol. 24:
138―142.
Crohn’s disease. Gastroenterology 115: 1405
14) De Man, J.C., Rogosa, M., and Sharpe, M.E. 1960. A medi-
―
1413.
29) Kucharzik, T., Lugering, N., Winde, G., Domschke, W.,
and Stoll, R. 1997. Colon carcinoma cell lines stimulate

IMMUNOREGULATORY CD14low MONOCYTES IN IEC CO-CULTURES
205
monocytes and lamina propria mononuclear cells to pro-
duce IL-10. Clin. Exp. Immunol. 110: 296 302.
30) Locher, C., Vanham, G., Kestens, L., Kruger, M., Ceup-
pens, J.L., Vingerhoets, J., and Gigase, P. 1994. Expression
patterns of Fc gamma receptors, HLA-DR and selected
adhesion molecules on monocytes from normal and HIV-
blood monocytes. Eur. J. Immunol. 26: 922―927.
45) Qiao, L., Schurmann, G., Betzler, M., and Meuer, S.C. 1991.
Activation and signaling status of human lamina propria T
―
lymphocytes. Gastroenterology 101: 1529―1536.
46) Rogler, G., Hausmann, M., Vogl, D., Aschenbrenner, E.,
Andus, T., Falk, W., Andreesen, R., Scholmerich, J., and
Gross, V. 1998. Isolation and phenotypic characterization of
infected individuals. Clin. Exp. Immunol. 98: 115―122.
31) MacDonald, T.T. 1999. Effector and regulatory lymphoid
cells and cytokines in mucosal sites. Curr. Top. Microbiol.
colonic macrophages. Clin. Exp. Immunol. 112: 205―215.
47) Rugtveit, J., Bakka, A., and Brandtzaeg, P. 1997. Differential
distribution of B7.1 (CD80) and B7.2 (CD86) costimulato-
ry molecules on mucosal macrophage subsets in human
inflammatory bowel disease (IBD). Clin. Exp. Immunol.
Immunol. 236: 113―135.
32) Macpherson, A.J., Gatto, D., Sainsbury, E., Harriman, G.R.,
Hengartner, H., and Zinkernagel, R.M. 2000. A primitive T
cell-independent mechanism of intestinal mucosal IgA
110: 104―113.
responses to commensal bacteria. Science 288: 2222
―
2226.
48) Rugtveit, J., Brandtzaeg, P., Halstensen, T.S., Fausa, O.,
and Scott, H. 1994. Increased macrophage subset in inflam-
matory bowel disease: apparent recruitment from peripher-
33) Malizia, G., Calabrese, A., Cottone, M., Raimondo, M.,
Trejdosiewicz, L.K., Smart, C.J., Oliva, L., and Pagliaro, L.
1991. Expression of leukocyte adhesion molecules by muco-
sal mononuclear phagocytes in inflammatory bowel dis-
al blood monocytes. Gut 35: 669―674.
49) Rugtveit, J., Haraldsen, G., Hogasen, A.K., Bakka, A.,
Brandtzaeg, P., and Scott, H. 1995. Respiratory burst of
intestinal macrophages in inflammatory bowel disease is
ease. Gastroenterology 100: 150―159.
34) McCormick, B.A., Colgan, S.P., Delp-Archer, C., Miller,
S.I., and Madara, J.L. 1993. Salmonella typhimurium attach-
ment to human intestinal epithelial monolayers: transcellu-
lar signalling to subepithelial neutrophils. J. Cell Biol. 123:
＋
＋
mainly caused by CD14 L1 monocyte derived cells. Gut
37: 367 373.
―
50) Rugtveit, J., Nilsen, E.M., Bakka, A., Carlsen, H., Brandtzaeg,
P., and Scott, H. 1997. Cytokine profiles differ in newly
recruited and resident subsets of mucosal macrophages from
895―907.
35) Moore, K.W., O’Garra, A., de Waal, M., Vieira, P., and
Mosmann, T.R. 1993. Interleukin-10. Annu. Rev. Immunol.
inflammatory bowel disease. Gastroenterology 112: 1493―
11: 165
―190.
1505.
36) Nathens, A.B., Rotstein, O.D., Dackiw, A.P., and Marshall,
J.C. 1995. Intestinal epithelial cells down-regulate
macrophage tumor necrosis factor-alpha secretion: a mech-
anism for immune homeostasis in the gut-associated lym-
51) Sartor, R.B. 1997. Pathogenesis and immune mechanisms of
chronic inflammatory bowel disease. Am. J. Gastroenterol.
92: 5S-11S.
52) Schreiber, S., Heinig, T., Thiele, H.G., and Raedler, A.
1995. Immunoregulatory role of interleukin-10 in patients
with inflammatory bowel disease. Gastroenterology 108:
phoid tissue. Surgery 118: 343―350.
37) Newberry, R.D., Stenson, W.F., and Lorenz, R.G. 1999.
Cyclooxygenase-2-dependent arachidonic acid metabolites
are essential modulators of the intestinal immune response to
dietary antigen. Nature 5: 900―906.
38) Oehler, J.R., and Herberman, R.B. 1977. Interaction of T
cells with allogeneic macrophages in mixed leukocyte cul-
1434―1444.
53) Schreiber, S., MacDermott, R.P., Raedler, A., Pinnau, R.,
Bertovich, M.J., and Nash, G.S. 1991. Increased activation of
isolated intestinal lamina propria mononuclear cells in
inflammatory bowel disease. Gastroenterology 101: 1020
―
tures in rats. J. Immunol. 119: 1785
―1789.
1030.
39) Panja, A., Goldberg, S., Eckmann, L., Krishen, P., and
Mayer, L. 1998. The regulation and functional consequence
of proinflammatory cytokine binding on human intestinal
54) Schreiber, S., Raedler, A., Conn, A.R., Rombeau, J.L., and
MacDermott, R.P. 1992. Increased in vitro release of soluble
interleukin-2 receptor by colonic lamina propria mononuclear
epithelial cells. J. Immunol. 161: 3675
―3684.
cells in inflammatory bowel disease. Gut 33: 236―241.
40) Pavli, P., Maxwell, L., Van de Pol, E., and Doe, F. 1996. Dis-
tribution of human colonic dendritic cells and macrophages.
55) Schwab, J.H. 1993. Phlogistic properties of peptidoglycan-
polysaccharide polymers from cell walls of pathogenic and
normal-flora bacteria which colonize humans. Infect. Immun.
Clin. Exp. Immunol. 104: 124―132.
41) Phalipon, A., and Sansonetti, P.J. 1999. Microbial-host inter-
actions at mucosal sites. Host response to pathogenic bacteria
61: 4535―4539.
56) Spöttl, T., Hausmann, M., Kreutz, M., Peuker, A., Vogl, D.,
Schölmerich, J., Falk, W., Andreesen, R., Andus, T., Herfarth,
H., and Rogler, G. 2001. Monocyte differentiation in intes-
tine-like macrophage phenotype induced by epithelial cells.
at mucosal sites. Curr. Top. Microbiol. Immunol. 236: 163
―
189.
42) Pullman, W.E., Elsbury, S., Kobayashi, M., Hapel, A.J., and
Doe, W.F. 1992. Enhanced mucosal cytokine production in
J. Leukoc. Biol. 70: 241―251.
inflammatory bowel disease. Gastroenterology 102: 529
537.
―
57) Sundberg, J.P., Elson, C.O., Bedigian, H., and Birkenmeier,
E.H. 1994. Spontaneous, heritable colitis in a new substrain
43) Qiao, L., Schurmann, G., Autschbach, F., Wallich, R., and
Meuer, S.C. 1993. Human intestinal mucosa alters T-cell
of C3H/HeJ mice. Gastroenterology 107: 1726―1735.
58) Targan, S.R., Deem, R.L., Liu, M., Wang, S., and Nel, A.
1995. Definition of a lamina propria T cell responsive state.
Enhanced cytokine responsiveness of T cells stimulated
reactivities. Gastroenterology 105: 814―819.
44) Qiao, L., Braunstein, J., Golling, M., Schurmann, G.,
Autschbach, F., Moller, P., and Meuer, S. 1996. Differential
regulation of human T cell responsiveness by mucosal versus
through the CD2 pathway. J. Immunol. 154: 664―675.