INFECTION AND IMMUNITY, May 2006, p. 2697–2705
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 5
Campylobacter jejuni Induces Maturation and Cytokine
Production in Human Dendritic Cells
Lan Hu, Mechelle D. Bray, Manuel Osorio, and Dennis J. Kopecko*
Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and
Drug Administration, 29 Lincoln Drive, NIH Campus, Building 29/420, HFM440, Bethesda, Maryland 20892
Received 28 October 2005/Returned for modification 27 November 2005/Accepted 1 February 2006
Campylobacter jejuni is a leading bacterial cause of human diarrheal disease in both developed and devel-
oping nations. Colonic mucosal invasion and the resulting host inflammatory responses are thought to be the
key contributing factors to the dysenteric form of this disease. Dendritic cells (DCs) play an important role in
both the innate and adaptive immune responses to microbial infection. In this study, the interaction between
human monocyte-derived dendritic cells and C. jejuni was studied. We found that C. jejuni was readily
internalized by DCs over a 2-h period. However, after a prolonged infection period (24 or 48 h) with C. jejuni,
only a few viable bacteria remained intracellularly. Minimal cytotoxicity of C. jejuni to dendritic cells was
observed. C. jejuni induced the maturation of dendritic cells over 24 h, as indicated by up-regulation of cell
surface marker proteins CD40, CD80, and CD86. In addition, Campylobacter-infected DCs triggered activation
of NF-?B and significantly stimulated production of interleukin-1? (IL-1?), IL-6, IL-8, IL-10, IL-12, gamma
interferon, and tumor necrosis factor alpha (TNF-?) compared to uninfected DCs. Active bacterial invasion of
DCs was not necessary for the induction of these cytokines, as heat-killed C. jejuni stimulated similar levels of
cytokine production as live bacteria. Purified lipooligosaccharide of C. jejuni appears to be the major stimulant
for the increased production of cytokines by DCs. Taken together, these data indicate that during infection,
Campylobacter triggers an innate inflammatory response through increased production of IL-1?, IL-6, IL-8,
and TNF-? and initiates a Th1-polarized adaptive immune response as predicted from the high level of
production of IL-12.
Campylobacter jejuni is a spiral, gram-negative, microaero-
philic bacterium. This pathogen is a leading bacterial cause of
human diarrheal disease throughout the world. In the United
States, about 2 million cases of diarrhea per year are caused by
C. jejuni. Illness caused by C. jejuni is characterized by fever,
headache, abdominal cramping, diarrhea, and the presence of
erythrocytes and leukocytes in the stool (4, 5). Two to 3 weeks
postinfection, C. jejuni is sometimes associated with the ex-
traintestinal manifestations of Guillain-Barre ´ syndrome or re-
active arthritis (43). The pathogenic mechanisms of C. jejuni
are not yet well understood. In developing countries, C. jejuni
triggers primarily a noninflammatory, watery diarrhea. How-
ever, in developed countries infection via bacterial invasion of
the colonic mucosa is thought to lead to intestinal inflamma-
tion and microulceration of the colonic mucosa. As is the case
with shigellosis, the acute host inflammatory responses are
Dendritic cells (DCs) play important roles in both the innate
and adaptive immune responses to microbial pathogens. They
are the major antigen-presenting cells (APCs) and are widely
distributed in tissues, including the intestinal mucosa (re-
viewed in references 26, 32, 53). Dendritic cells regulate the
type of T-cell-mediated immune response to an offending
agent and are also a source of proinflammatory cytokines.
They are the only cells that are able to initiate proliferation in
naı ¨ve T cells, thereby inducing the primary immune response
and permitting the establishment of immunological memory.
Immature DCs can capture antigens by phagocytosis, mac-
ropinocytosis, and endocytosis (17, 37, 38). Exposure of DCs to
inflammatory stimuli converts these cells from antigen-captur-
ing immature DCs to antigen-presenting mature DCs. This
process is accompanied by up-regulation of major histocom-
patibility complex (MHC) class I and II molecules, costimula-
tory receptors, and adhesion molecules (2, 7, 11). Once a DC
has captured an antigen, its ability to capture additional anti-
gens rapidly declines. The process of differentiation from an
immature DC into a mature professional APC can be induced
by whole bacteria, their components, or cytokines as well as
other inflammatory stimuli and infectious agents. Upon acti-
vation, DCs migrate from the inflammatory site into the lym-
phoid tissues, such as the lymph nodes and spleen, down-
regulate their phagocytic ability, and up-regulate their antigen-
presenting capacity (2).
Numerous studies have shown that DCs play an important
role in the host-pathogen interaction during infection with
enteric pathogenic bacteria. For example, DCs that phagocy-
tose Salmonella enterica serovar Typhimurium can process
and present bacterial antigens and produce cytokines that
are critical to the immune response (25, 56). Helicobacter
pylori also induces maturation and cytokine release from
human DCs (24). Shigella flexneri infection of DCs leads to
up-regulation of interleukin-1? (IL-1?) and IL-18 and rapid
DC death (8). In contrast, Yersinia enterocolitica is able to
invade DCs and does not induce necrosis or apoptosis but
impairs DC function (40).
* Corresponding author. Mailing address: Laboratory of Enteric and
Sexually Transmitted Diseases, Center for Biologics Evaluation and
Research, Food and Drug Administration, 29 Lincoln Drive, NIH
Campus, Bldg. 29/420, HFM440, Bethesda, MD 20892. Phone: (301)
496-1893. Fax: (301) 402-8701. E-mail: firstname.lastname@example.org.
During infection, Campylobacter can invade and traverse
the epithelial barrier and comes into contact with numerous
leukocytes (49, 50). In addition, DCs are capable of travers-
ing the tight junctions of the intestinal mucosa, which en-
ables them to interact directly with bacteria on the mucosal
surface (35, 39). Although the infection of macrophages by
C. jejuni has been studied previously (14, 19, 20, 42, 44, 51),
Campylobacter’s interaction with DCs has not been reported
to date. Understanding the interaction between DCs and C.
jejuni will provide insight into the role of DCs in stimulating
Campylobacter-induced inflammatory pathology and in con-
trolling the infection. The goal of this work was to study the
interaction between human DCs and C. jejuni with respect to
DC activation and induction of a number of cytokines known
to be involved in both the innate and adaptive immune re-
MATERIALS AND METHODS
Bacterial strains and culture conditions. Campylobacter jejuni strain 81-176,
obtained following a disease outbreak in Minnesota, has been shown to cause
colitis in two human challenge studies (4, 23; D. Tribble et al., unpublished data).
C. jejuni 81-176 and its mutant RY213 (a cheY?diploid) that is noninvasive (55),
as well as the genome-sequenced C. jejuni NCTC 11168 (33), were grown in
Mueller-Hinton (M-H) biphasic broth or on M-H agar (Difco) at 37°C under a
Campylobacter gas atmosphere of 10% carbon dioxide, 5% oxygen, and 85%
Preparation of human monocyte-derived dendritic cells. Mononuclear cells
were obtained by apheresis of normal volunteer donors as performed by the
Blood Services Section of the Department of Transfusion Medicine at the Na-
tional Institutes of Health Warren G. Magnuson Clinical Center (Bethesda,
MD). The mononuclear cells were further enriched for monocytes by centrif-
ugal elutriation as performed by the Cell Processing Section of the Depart-
ment of Transfusion Medicine. Similar to the work of Pickering et al. (34), the
elutriated monocytes were then cultured (1 ? 106/well) in six-well tissue culture
plates with 3 ml/well of RPMI 1640 medium (Mediatech, Inc., Herndon, VA)
containing 5% human AB serum (Nabi, Miami, FL), 800 U/ml granulocyte-macro-
phage colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, NJ), and 500
U/ml IL-4 (Peprotech). The nonadherent and loosely adherent cells were harvested
after incubating the plates for 6 days at 37°C in 5% CO2. The cells were centrifuged
for 10 min at 1,200 rpm in a Beckman GS-6KR centrifuge and then plated in 96-
(50,000 cells/well) or 6-well plates (106cells/well) containing fresh RPMI (Me-
diatech) with 5% human AB serum (Nabi) and 800 U/ml GM-CSF (Peprotech).
This resulted in the generation of immature monocyte-derived dendritic cells
that were positive for CD11c but negative for CD14 (data not shown).
Infection of DCs. C. jejuni was added to the cultured DCs at different multi-
plicities of infection (MOIs). Bacterium-DC interactions were initiated by cen-
trifugation at 1,000 rpm in a Beckman GS-6KR centrifuge for 5 min, followed by
incubation at 37°C in 5% CO2. For assessment of the number of intracellular
bacteria, 2 ? 105infected DCs/well in 24-well plates were cultured for 2, 4, 24,
and 48 h. After the 4-h time point, gentamicin (20 ?g/ml) was added to inhibit
the growth of extracellular bacteria (19). At the specified time points (2, 4, 24,
and 48 h), cells were washed three times with RPMI 1640, followed by incubation
of DCs for 2 h in fresh medium including 100 ?g/ml gentamicin to kill any
remaining extracellular bacteria. The infected monolayers were then washed
three times to remove the gentamicin. The DCs were lysed with 0.1% Triton
X-100 in phosphate-buffered saline (PBS) for 15 min. Following serial dilutions
in PBS, the viable internalized bacteria were enumerated by plate count on M-H
agar. For cytokine measurements, the infected culture supernatants in 96-well
plates were harvested, centrifuged, and frozen at ?80°C prior to analysis (12, 34).
In control studies, 100 ?g/ml gentamicin was found to kill all extracellular
bacteria within 2 h. The MIC of gentamicin at which 50% of the C. jejuni
81-176 organisms were inhibited was ?6.25 ?g/ml for C. jejuni 81-176. At 20
?g/ml of gentamicin, we found that extracellular C. jejuni could not multiply
in cell culture medium. We used 20 ?g/ml gentamicin to block any extracel-
lular growth of released C. jejuni over longer periods (i.e., 24 to 48 h),
because higher antibiotic concentrations over long periods can leak into the
host cells and cause death of some intracellular bacteria.
Cell cytotoxicity assay. The CytoTox 96 assay (Promega, Madison, WI) was
used to determine host cell cytotoxicity over time induced by bacterial prepara-
tions that were added to the DCs. This assay qualitatively measures supernatant
lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon
cell lysis. The DC number was evaluated via the assay instructions and deter-
mined to be optimal at 50,000 cells/well. The ratios of effector cells to bacteria
used to assess host cell cytotoxicity were 1:1, 1:10, 1:20, and 1:100. Superna-
tants were collected following centrifugation at 4, 24, and 48 h postinfection
with C. jejuni 81-176. Maximum LDH release was determined by measuring
the amount of LDH release from uninfected DCs that were treated with lysis
buffer. The percentage of cytotoxicity was calculated according to the fol-
lowing formula (OD is an abbreviation for optical density): [(ODsample) ?
(ODmedium/ODmax LDH release) ? ODadjusted medium] ? 100.
Cytokine measurement of DC supernatants by Luminex assay. Immature DCs
were incubated in 96-well plates (5 ? 104DCs/well) for 4, 24, and 48 h with live
C. jejuni 81-176, heat-killed (i.e., 70°C for 30 min) C. jejuni 81-176, lipooligosac-
charide (LOS) of C. jejuni 81-176 (extracted by using the hot phenol-water
technique ), or hot phenol-water-extracted lipopolysaccharide (LPS) of Esch-
erichia coli (Sigma, St. Louis, MO). PBS (Invitrogen Corporation)-incubated
DCs served as unstimulated controls. Cytokine assays were performed by using
human cytokine 10-plex and IL-12p70 antibody bead kits (Biosource Interna-
tional, Camarillo, CA) and a Luminex 100 analyzer (Luminex Corporation,
Austin, TX) for IL-1?, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, gamma inter-
feron (IFN-?), tumor necrosis factor alpha (TNF-?), and GM-CSF. Cytokine
data analysis was performed using the MasterPlex QT quantitation software
(MiraiBio, Alameda, CA).
Flow cytometry. Flow cytometric analysis was performed in order to examine
the cell surface maturation markers CD40, CD80, and CD86 in Campylobacter-
infected DCs. Dendritic cells were infected as described above. Maturation of
DCs was determined at 24 and 48 h after infection with C. jejuni (MOI, 10) by
checking the expression of the surface maturation markers by flow cytometry.
Cell samples (5 ? 105cells) were incubated (20 min, 4°C) with 4% human AB
serum (Nabi) in PBS (Invitrogen Corporation) to block nonspecific binding sites
and Fc receptors. The cells were washed via centrifugation (1,200 rpm; 10 min)
with fluorescence-activated cell sorter buffer containing 0.2% bovine serum al-
bumin (Sigma) and 0.1% sodium azide (Sigma) in PBS (Invitrogen Corporation)
and then incubated (45 min, 4°C) with anti-CD40 (5C3), anti-CD 80 (L307.4), or
anti-CD86 (2331 [FUN-1]) fluorescein isothiocyanate-labeled antibody (1 ?l;
Becton Dickinson, San Jose, CA) or the isotype-matched (1 ?l of immunoglob-
ulin G1; X40) control antibody (Becton Dickinson). After washing again, the
cells were resuspended in 0.5% paraformaldehyde (Sigma) in cold PBS (Invitro-
gen) and 0.1% sodium azide. The cell-associated immunofluorescence was mea-
sured with a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry
Systems, San Jose, CA) and analyzed using CellQuest software (B-D Bio-
NF-?B transcription factor assay. The TransAM NF-?B kit p65 (Active Motif,
Carlsbad, CA) was employed to detect the activation of NF-?B. At 4 h post-
infection, the nuclear extracts were prepared as per the manufacturer’s instruc-
tions. Protein concentrations were determined using a 2?-benzoyloxycinnamal-
dehyde protein assay reagent kit (Pierce, IL). Nuclear extracts (10 ?g/ml) were
added into each well of a 96-well plate and incubated with anti-p65, and NF-?B
concentrations were determined spectrophotometrically per the kit instructions.
Statistical analysis. Results are presented as the mean ? standard error of the
mean from three independently conducted assays. P values were calculated with
Student’s t test.
Viable intracellular bacteria in dendritic cells after C. jejuni
infection. C. jejuni was added to DCs at MOIs of 1, 10, 20, and
100. After a 2-, 4-, 24-, or 48-h invasion period, followed by a
2-h gentamicin (100 ?g/ml) treatment to kill all extracellular
bacteria, the numbers of viable, intracellular Campylobacter
were quantified by plate count after host cell lysis. At 2 and 4 h
postinfection, the uptake was generally dose dependent, and
higher doses of bacteria led to greater levels of bacterial inter-
nalization by DCs (Fig. 1). The average number of viable
intracellular bacteria per DC was ?1.5 bacteria/DC on average
at 2 or 4 h at an MOI of 10 and increased about fourfold at an
MOI of 100. By 24 or 48 h postinfection, however, the numbers
2698 HU ET AL.INFECT. IMMUN.
of viable intracellular bacteria were dramatically reduced. For
example, at an MOI of 100, only ?1% of DCs contained one
viable bacterium, with even less observed at 48 h. These data
suggest that C. jejuni is internalized at early time points, but
most intracellular bacteria are killed by 24 and 48 h postinfec-
tion. This assumption is predicated on little or no cytotoxicity
to DCs during bacterial infection, which was studied next.
DC viability after infection with C. jejuni. To assess the
viability over time of DCs following infection with C. jejuni,
LDH release assays were performed using the supernatants of
both uninfected and infected cells. No significant differences in
DC viability were detected at 4 and 24 h after infection at any
bacterial MOI, in comparison to the uninfected controls (Fig.
2). At 48 h postinfection, all infected DCs showed a minimal
increase in cytotoxicity over the uninfected control. However,
the 9.5% dead cells in the uninfected control at 48 h was only
doubled to ?20% death in the infected DCs at the highest
MOI. These data suggest that limited host cell cytotoxicity was
associated with both long infection times and higher MOIs.
However, infection-induced DC death over 48 h was very lim-
ited even at the highest MOI.
C. jejuni effects on DC maturation. To study the effects on
DC maturation of Campylobacter infection, the expression
of cell surface costimulatory molecules CD40, CD80, and
CD86 was measured with flow cytometry. Compared to un-
infected cultures, C. jejuni-infected DCs showed increased
expression of all three surface molecules at 24 h postinfec-
tion, as indicated by increased fluorescence intensity of DCs
(Fig. 3). The averaged mean fluorescence intensity measure-
ments for CD40, CD80, and CD86 at 24 h in all three sets of
control DCs were 12.52 ? 0.99, 7.01 ? 0.38, and 45.28 ? 10.83,
respectively. Infection with C. jejuni 81-176 significantly in-
creased these maturation marker averaged mean fluorescence
intensity measurements to 21.66 ? 3.84 (P ? 0.05), 25.99 ?
10.83 (P ? 0.01), and 96.16 ? 7.35 (P ? 0.005), respectively. As
a positive control, purified E. coli LPS induced DC maturation
similarly to that observed with live C. jejuni (data not shown).
These data show that C. jejuni infection stimulates an increase
in the expression of DC surface costimulatory molecules,
which are indicators of DC maturation.
C. jejuni infection induces the production of cytokines in
human DCs. The kinetics and profile of cytokine secretion from
human DCs during Campylobacter infection were analyzed. Cell
culture supernatants were collected at 4, 24, and 48 h from unin-
fected DCs or after infection with strain 81-176 at different MOIs,
and cytokine levels were measured in multiplex bead assays.
Campylobacter infection of DCs induced enhanced production of
IL-2, IL-4, or IL-5 (data not shown).
At 4 h postinfection with C. jejuni at an MOI of 10, the titers
of IL-6, IL-8, and TNF-? were increased ?29-fold (837.80 ?
83.00 pg/ml), 34-fold (1,073.70 ? 282.90 pg/ml), and 1,959-fold
(46,596.30 ? 11,977.78 pg/ml), respectively, compared to the
uninfected controls (Fig. 4B, C, and G). The production of
IL-6 and IL-8 as well as IL-12 and IFN-? (Fig. 4E and F) was
significantly increased at 24 and 48 h postinfection in compar-
ison to the 4-h time point. TNF-? was detected at high levels
at all time points from infected DCs. C. jejuni 81-176 induced
comparatively lower quantities of IL-1? or IFN-? than the
other cytokines; however, IL-1? and IFN-? levels were signif-
icantly increased at 24 and 48 h postinfection relative to the 4-h
time point (Fig. 4A and F). Finally, substantial increases in
IL-10 were only observed at the 48-h time point (Fig. 4D).
These data show that increases in the Campylobacter MOI
from 10 to 100 had little impact on overall cytokine production.
Cytokine production increased over time, with peak amounts
at 24 or 48 h.
FIG. 1. Measurement of viable intracellular bacteria in dendritic
cells after C. jejuni infection. C. jejuni was added to DCs at MOIs of 1,
10, 20, and 100. After various invasion periods, the cells were washed
three times, followed by an incubation of the DCs for 2 h in fresh
medium containing 100 ?g/ml gentamicin to kill any remaining extra-
cellular bacteria. The data represent the mean ? standard deviation of
duplicate wells from three independent assays. *, the number of viable
intracellular bacteria had markedly decreased at 24 to 48 h and does
not appear on this graph.
FIG. 2. DC viability after infection with C. jejuni 81-176. LDH in
the supernatants of infected and uninfected DCs was sampled and
measured at 4, 24, and 48 h after infection at various MOIs. The data
are presented as means ? standard errors of the means from three
separate experiments. The values are expressed as percent host cell
cytotoxicity, relative to the uninfected cell control obtained by lysing
uninfected DCs. The insert shows the data on an expanded scale for
VOL. 74, 2006 C. JEJUNI INDUCES DC MATURATION AND CYTOKINE RELEASE 2699
Is active bacterial invasion or bacterial viability necessary to
induce cytokine production in DCs? Wild-type invasive C. jejuni
81-176, the noninvasive RY213 mutant strain of 81-176 (55),
NCTC 11168, a minimally invasive C. jejuni strain (1), and heat-
killed 81-176 were compared for their ability to trigger DC secre-
tion of selected cytokines. C. jejuni 81-176 was heat treated at
70°C for 30 min and subsequently shown by plate count to be
nonviable. The production levels of IL-1?, IL-6, IL-8, IFN-?, and
for all strains. The mutant RY213 and NCTC 11168 showed
slightly lower IL-10 and IL-12 induction levels compared to live
slightly lower cytokine levels than live 81-176 cells, but this dif-
ference was, in most cases, not significant. Using an MOI of 100,
the heat-killed bacteria did not induce any higher cytokine levels
than when at an MOI of 20 (data not shown). These data suggest
that neither active bacterial invasion of DCs nor bacterial viability
is necessary for C. jejuni to induce cytokine production, but live
wild-type 81-176 cells generally result in slightly better induction
than other stimulants.
Comparing polysaccharides of C. jejuni and E. coli for ability
to induce cytokines in DCs. LOS was prepared from C. jejuni
81-176 by the hot phenol-water extraction method and identi-
fied as LOS on silver-stained sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis gels (data not shown). LOS
(100 ng/ml) was added to DCs for 24 h. An equal amount of
purified LPS from E. coli, known to induce cytokine produc-
tion in DCs, was used as a control. Both LOS and LPS induced
cytokine levels similar to that seen with live C. jejuni (Fig. 6), with
the exception of a lower IL-10 induction by C. jejuni LOS. These
data suggest that LOS of C. jejuni is a key contributor to the
induction of cytokine production by DCs.
NF-?B activation after infection of DCs with C. jejuni. The host
transcription factor NF-?B promotes transcription of various im-
FIG. 3. Surface molecules of DCs as determined by flow cytometry 24 h after exposure to C. jejuni 81-176. The DCs were stained with
fluorescence-conjugated monoclonal antibodies to human CD40, CD80, and CD86. Histograms depict the level of surface expression, and the value
indicated on each histogram is the mean fluorescence intensity of the marker-specific antibody from one representative experiment of three
different donors. Black histograms indicate the levels of fluorescence using isotype control antibodies, and the open histograms represent the cell
surface molecule expression in infected or uninfected DCs.
2700 HU ET AL.INFECT. IMMUN.
FIG. 4. Cytokine induction in human DCs by C. jejuni 81-176 at different MOIs. Culture supernatants were tested at 4, 24, and 48 h
postinfection with C. jejuni 81-176 at an MOI of 10, 20, or 100 and compared to that of uninfected DCs. The release of IL-1?, IL-6, IL-8, IL-10,
IL-12, IFN-?, and TNF-? is shown (A, B, C, D, E, F, and G, respectively). Data are presented as the mean ? standard deviation of duplicate wells
from three independent assays.
VOL. 74, 2006 C. JEJUNI INDUCES DC MATURATION AND CYTOKINE RELEASE2701
mune response genes, including cytokines (10). The nuclear ex-
tracts exhibited low NF-?B activation in the uninfected control
in DCs infected with C. jejuni 81-176 for 4 h (Fig. 7).
DCs play important roles in both the innate and adaptive
immune responses to microbial pathogens (26). The intestinal
FIG. 5. Comparison of cytokine levels induced in DCs infected by live invasive, noninvasive, or heat-killed strain 81-176. Culture supernatants were
tested 24 h postinfection with C. jejuni at an MOI of 20. The release of IL-1?, IL-6, IL-8, IL-10, IL-12, IFN-?, and TNF-? is shown (A, B, C, D, E, F,
and G, respectively). Data are presented as the mean ? standard deviation of duplicate wells from three independent assays.
2702HU ET AL.INFECT. IMMUN.
immune system DCs lie beneath the mucosal surface in Peyer’s
patches and form an early line of defense against invading
pathogens. Intestinal DCs can internalize bacterial pathogens
following their uptake through M cells or more directly
through paracellular dendrite extensions into the intestinal
lumen (35). Here, we analyzed the interaction between C.
jejuni and human DCs to investigate DC activation, potential
bacterial or DC toxicity, and the induction of cytokines asso-
ciated with disease inflammation and immune responses to
infection. Our data show that DCs internalize C. jejuni at early
time points (i.e., 2 h postinfection), taking up one to six bac-
teria per DC on average at MOIs of 10 to 100. Ninety-nine
percent of internalized C. jejuni were killed within 24 h, and
there was virtually no cytotoxicity to DCs induced over 24 h,
with only minimal increased DC death (?10%) after 48 h.
Wassenaar et al. (51) found that activated human monocytes/
macrophages also efficiently killed C. jejuni, causing a 5-log
reduction in 24 h. Our results differ from those of Siegesmund
et al. (42), who reported that C. jejuni F38011 induced apop-
tosis in 63% of THP-1 (human monocyte line) cells 48 h after
In striking contrast, many studies report that Salmonella is
internalized by and survives within both human and murine
DCs (6, 9, 18, 29, 45), although Salmonella has been reported
to rapidly kill infected DCs and macrophages (21, 25, 48).
Shigella infection of human DCs results in rapid IpaB-depen-
dent DC death (8), which probably dampens the adaptive im-
mune response to this enteric pathogen. Similar to our findings
with Campylobacter, Y. enterocolitica enters DCs and does not
induce necrosis or apoptosis (40). However, Yersinia reverses
the activation of DCs, reduces DCs’ abilities to stimulate an
adaptive response, and prolongs infection. For Campylobacter
and some enteric pathogens, it is still largely unclear as to what
primary factor(s) determines intracellular bacterial survival or
triggers host cell necrosis/apoptosis.
In this study, C. jejuni stimulation of immature DCs resulted
in their maturation to APCs, as evidenced by enhanced expres-
sion of cell surface costimulatory molecules CD40, CD80, and
CD86. Similarly, other enteric pathogens, such as S. enterica
serovar Typhimurium (30) or Helicobacter pylori (24), have
been shown to up-regulate DC surface expression of MHC
class II, CD40, CD80, and CD86 molecules. Coordinated up-
regulation of the costimulatory molecules and translocation of
MHC molecules to the cell surface are essential molecular
events for subsequent antigen presentation and ultimate acti-
vation of both CD4?and CD8?T cells (36).
Monocytes/macrophages are considered early host respond-
ers to infection and are also an important source of proinflam-
matory cytokines. C. jejuni has recently been reported to in-
duce IL-1, IL-6, IL-8, and TNF-? in human monocytes (14,
19). Dendritic cells also produce various cytokines and chemo-
kines upon interaction with pathogenic bacteria (8, 24, 25, 56).
Our data show that C. jejuni activates the transcriptional factor
FIG. 6. Comparison of the potential of C. jejuni LOS and E. coli
LPS to induce cytokine production in human DCs. LOS (100 ng/ml)
from C. jejuni and LPS (100 ng/ml) from E. coli were added to DCs for
24 h. The release of IL-6, IL-8, and TNF-? (A) or IL-1?, IL-10, and
IFN-? (B) are compared to values for uninfected DCs (due to the
scale, uninfected DC data are too low to be visible in panel A). Data
are presented as the mean ? standard deviation of duplicate wells
from three independent assays.
FIG. 7. C. jejuni-induced activation of NF-?B in DCs. DCs were
incubated with C. jejuni 81-176 for 4 h. Nuclear extracts were prepared
and incubated with anti-NF-?B p65 per the instructions of the manu-
facturer. The NF-?B concentrations were determined spectrophoto-
metrically. The data represent the mean ? standard deviation from
three independent assays.
VOL. 74, 2006C. JEJUNI INDUCES DC MATURATION AND CYTOKINE RELEASE 2703
NF-?B and induces the production of significant amounts of
IL-1?, IL-6, IL-8, IL-10, IL-12, IFN-?, and TNF-? in DCs.
These cytokines play crucial roles in the induction of inflam-
mation and in adaptive immune responses (28, 31, 47).
Proinflammatory cytokines IL-1?, IL-6, IL-8, and TNF-?
were induced rapidly in DCs infected with C. jejuni and were
secreted and maintained at high levels over 48 h. The cytokine
expression pattern was not markedly altered by increasing the
MOI from 10 to 100. These findings add to the growing body
of information which shows that C. jejuni triggers the innate
immune system to produce proinflammatory cytokines, which
likely serve to initiate and modulate local inflammation in the
Peyer’s patches, leading to disease symptoms. Previous studies
of C. jejuni-infected human cell lines have shown that this
pathogen also induces epithelial cell secretion of IL-8 and
some chemokines (13, 15, 27), attracting phagocytic cells that
may be important in both containing and in exacerbating the
infection (54). Consistent with these results, our studies show
that many proinflammatory cytokines, including IL-8, were
stimulated in a significant and rapid fashion in DCs.
As DCs mature, they synthesize cytokines essential for the
development of T-cell interactions (i.e., adaptive responses).
Compared to other types of APCs, DCs are 1,000-fold more
efficient in activating resting T cells (3). Interleukin-12 appears
to be a key cytokine produced by APCs to stimulate a Th1-
directed response (7, 46). C. jejuni induced very high levels of
IL-12 at 24 and 48 h after infection. IL-10 is an important
Th2-type cytokine that up-regulates humoral and down-regu-
lates cell-mediated immune responses (31, 41). The high IL-12
production (i.e., 10,000-fold induction) and relatively low
IL-10 induction (i.e., 100- to 500-fold induction) by C. jejuni
favors a Th1 response.
The induction of cytokine production in DCs was not signif-
icantly different between live and heat-killed bacteria, except
for IL-10, which was slightly lower with the heat-killed bacte-
rial stimulus. Similar findings were previously reported in C.
jejuni-infected monocytes (19). The noninvasive RY213 strain
and C. jejuni NCTC 11168, which have low invasion efficiencies
(1), induced cytokines IL-10, IL-12, and TNF-? at levels re-
duced only by about one-half versus strain 81-176. These data
argue that invasion is not necessary for the induction of these
cytokines. In fact, purified LOS from strain 81-176 induced
high levels of the selected cytokines studied, with the exception
of IL-10. These data indicate that the predominant induction
of cytokines in DCs may be due to interaction with LOS. Some
alternate pathway is likely needed for full IL-10 induction. The
signal transduction pathways modulated by the interaction of
C. jejuni with DCs are currently being investigated in our lab.
Taken together, these data suggest that DCs are an important
component of the host-pathogen interaction with Campy-
lobacter. Further, DCs play a role in triggering inflammatory
cytokines likely involved in disease pathogenesis and in initi-
ating a Th1-directed adaptive immune response during C. je-
We acknowledge T. T. Wai for help in preparing Campylobacter
LOS and thank R. I. Walker, M. Akkoyunlu, and S. Dharwan for
critical review of the manuscript.
1. Bacon, D. J., R. A. Alm, D. H. Burr, L. Hu, D. J. Kopecko, C. P. Ewing, T. J.
bacter jejuni 81–176. Infect. Immun. 68:4384–4390.
2. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of
immunity. Nature 392:245–252.
3. Bhardwaj, N., J. W. Young, A. J. Nisanian, J. Baggers, and R. M. Steinman.
1993. Small amounts of superantigen, when presented on dendritic cells, are
sufficient to initiate T cell responses. J. Exp. Med. 178:633–642.
4. Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughes, and M. J. Blaser.
1988. Experimental Campylobacter jejuni infection in humans. J. Infect. Dis.
5. Blaser, M. J. 1997. Epidemiologic and clinical features of Campylobacter
jejuni infections. J. Infect. Dis. 176(Suppl. 2):S103–S105.
6. Cano, D. A., M. Martinex-Moya, M. G. Pucciarelli, E. A. Groisman, J.
Casadesus, and F. Garcia-Del Portillo. 2001. Salmonella enterica serovar
Typhimurium response involved in attenuation of pathogen intracellular
proliferation. Infect. Immun. 69:6463–6474.
7. de Saint-Vis, B., I. Fugier-Vivier, C. Massacrier, C. Gaillard, B. Vanbervliet,
S. Ait-Yahia, J. Banchereau, Y.-J. Liu, S. Lebecque, and C. Caux. 1998. The
cytokine profile expressed by human dendritic cells is dependent on cell
subtype and mode of activation. J. Immunol. 160:1666–1676.
8. Edgeworth, J. D., J. Spencer, A. Phalipon, G. E. Griffin, and P. J. Sansonetti.
2002. Cytotoxicity and interleukin-1? processing following Shigella flexneri
infection of human monocyte-derived dendritic cells. Eur. J. Immunol. 32:
9. Garcia-Del Portillo, F., H. Jungnitz, M. Rohde, and C. A. Guzman. 2000.
Interaction of Salmonella enterica serotype Typhimurium with dendritic cells
is defined by targeting to compartments lacking lysosomal membrane glyco-
proteins. Infect. Immun. 68:2985–2991.
10. Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF-kappa B and Rel proteins:
evolutionarily conserved mediators of immune responses. Annu. Rev. Im-
11. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, and S. Amigorena.
2002. Antigen presentation and T cell stimulation by dendritic cells. Annu.
Rev. Immunol. 20:621–667.
12. Guiney, D. G., P. Hasegawa, and S. P. Cole. 2003. Helicobacter pylori pref-
erentially induces interleukin 12 (IL-12) rather than IL-6 or IL-10 in human
dendritic cells. Infect. Immun. 71:4163–4166.
13. Hickey, T. E., S. Baqar, L. Bourgeois, C. P. Wesing, and P. Guerry. 1999.
Campylobacter jejuni-stimulated secretion of interleukin-8 by INT407 cells.
Infect. Immun. 67:88–93.
14. Hickey, T. E., G. Majam, and P. Guerry. 2005. Intracellular survival of
Campylobacter jejuni in human monocytic cells and induction of apoptotic
death by cytolethal distending toxin. Infect. Immun. 73:5194–5197.
15. Hu, L., and T. E. Hickey. 2005. Campylobacter jejuni induces secretion of
proinflammatory chemokines from human intestinal epithelial cells. Infect.
16. Hu, L., and D. J. Kopecko. 1999. Campylobacter jejuni 81–176 associates with
microtubules and dynein during invasion of human intestinal cells. Infect.
17. Inaba, K., M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara, S.
Muramatsu, and R. M. Steinman. 1993. Granulocytes, macrophages, and
dendritic cells arise from a common major histocompatibility complex class
II-negative progenitor in mouse bone marrow. Proc. Natl. Acad. Sci. USA
18. Jantsch, J., C. Cheminay, D. Chakravortty, T. Lindig, J. Hein, and M.
Hensel. 2003. Intracellular activities of Salmonella enterica in murine den-
dritic cells. Cell. Microbiol. 5:933–945.
19. Jones, M. A., S. Totemeyer, D. J. Maskell, C. E. Bryant, and P. A. Barrow.
2003. Induction of proinflammatory responses in the human monocytic cell
line THP-1 by Campylobacter jejuni. Infect. Immun. 71:2626–2633.
20. Kielhbauch, J. A., R. A. Albach, L. L. Bauum, and K.-P. Chang. 1985.
Phagocytosis of Campylobacter jejuni and its intracellular survival in mono-
nuclear phagocytes. Infect. Immun. 48:446–451.
21. Knodler, L. A., and B. B. Finlay. 2001. Salmonella and apoptosis: to live or
let die? Microbes Infect. 3:1321–1326.
22. Kopecko, D. J., L. Hu, and K. J. Zaal. 2001. Campylobacter jejuni-micro-
tubule-dependent invasion. Trends Microbiol. 9:389–396.
23. Korlath, J. A., M. T. Osterholm, L. A. Judy, J. C. Forfang, and R. A.
Robinson. 1985. A point-source outbreak of campylobacteriosis associated
with consumption of raw milk. J. Infect. Dis. 152:592–596.
24. Kranzer, K., A. Eckhardt, M. Aigner, G. Knoll, L. Deml, C. Speth, N. Lehn,
M. Rehli, and W. Schneider-Brachert. 2004. Induction of maturation and
cytokine release of human dendritic cells by Helicobacter pylori. Infect. Im-
25. Marriott, I., T. G. Hammond, E. K. Thomas, and K. L. Bost. 1999. Salmo-
nella efficiently enter and survive within cultured CD11c?dendritic cells
initiating cytokine expression. Eur. J. Immunol. 29:1107–1115.
26. Medzhitov R., and C. A. Janeway, Jr. 1997. Innate immunity: the virtues of
a nonclonal system of recognition. Cell 91:295–298.
2704 HU ET AL.INFECT. IMMUN.
27. Mellits, K. H., J. Mullen, M. Wand, G. Armbruster, A. Patel, P. L. Connerton, Download full-text
M. Skelly, and I. F. Connerton. 2002. Activation of the transcription factor
NF-?B by Campylobacter jejuni. Microbiology 148:2753–2763.
28. Nicod, L. P., and J. M. Dayer. 1999. Cytokines in the functions of dendritic
cells, monocytes, macrophages and fibroblasts. Oxford University Press, New
29. Niedergang, F., J. C. Sirard, C. T. Blanc, and J. P. Kraehenbuhl. 2000. Entry
and survival of Salmonella typhimurium in dendritic cells and presentation of
recombinant antigens do not require macrophage-specific virulence factors.
Proc. Natl. Acad. Sci. USA 97:14650–14655.
30. Norimatsu, M., V. Chance, G. Dougan, C. J. Howard, and B. Villarreal-
Ramos. 2004. Live Salmonella enterica serovar Typhimurium (S. typhi-
murium) elicit dendritic cell responses that differ from those induced by
killed S. typhimurium. Vet. Immunol. Immunopathol. 98:193–201.
31. Opal, S. M., J. C. Wherry, and P. Grint. 1998. Interleukin-10: potential
benefits and possible risks in clinical infectious diseases. Clin. Infect. Dis.
32. Palucka, K., and J. Banchereau. 2002. How dendritic cells and microbes
interact to elicit or subvert protective immune responses. Curr. Opin. Im-
33. Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T.
Chillingworth, R. M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A. V. Karlyshev,
S. Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M.
Rutherford, A. H. van Vliet, S. Whitehead, and B. G. Barrell. 2000. The genome
sequence of the food-borne pathogen Campylobacter jejuni reveals hypervari-
able sequences. Nature 403:665–668.
34. Pickering, A. K., M. Osorio, G. M. Lee, V. K. Grippe, M. Bray, and T. J.
Merkel. 2004. Cytokine response to infection with Bacillus anthracis spores.
Infect. Immun. 72:6382–6389.
35. Rescigno, M., M. Urbano, B. Valzasina, M. Francolini, G. Rotta, R. Bonasio,
F. Granucci, J. P. Kraehenbuhl, and P. Ricciardi-Castagnoli. 2001. Den-
dritic cells express tight junction proteins and penetrate gut epithelial mono-
layers to sample bacteria. Nat. Immunol. 2:361–367.
36. Rescigno, M., M. Martino, C. L. Sutherland, M. R. Gold, and P. Ricciardi-
Castagnoli. 1998. Dendritic cell survival and maturation are regulated by
different signaling pathways. J. Exp. Med. 188:2175–2180.
37. Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995. Dendritic cells
use macropinocytosis and the mannose receptor to concentrate macromol-
ecules in the major histocompatibility complex class II compartment: down-
regulation by cytokines and bacterial products. J. Exp. Med. 182:389–400.
38. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble
antigen by cultured human dendritic cells is maintained by granulocyte/
macrophage colony-stimulating factor plus interleukin 4 and downregulated
by tumor necrosis factor alpha. J. Exp. Med. 179:1109–1118.
39. Scheinecker, C., R. McHugh, E. M. Shevach, and R. N. Germain. 2002.
Constitutive presentation of a natural tissue autoantigen exclusively by den-
dritic cells in the draining lymph node. J. Exp. Med. 196:1079–1090.
40. Schoppet, M., A. Bubert, and H. I. Huppertz. 2000. Dendritic cell function is
perturbed by Yersinia enterocolitica infection in vitro. Clin. Exp. Immunol.
41. Sharma, S., M. Stolina, Y. Lin, B. Gardner, P. W. Miller, M. Kronenberg,
and S. M. Dubinett. 1999. T cell-derived IL-10 promotes lung cancer growth
by suppressing both T cell and APC function. J. Immunol. 163:5020–5028.
42. Siegesmund, A. M., M. E. Konkel, J. D. Klena, and P. F. Mixter. 2004.
Campylobacter jejuni infection of differentiated THP-1 macrophages results
in interleukin 1 beta release and caspase-1-independent apoptosis. Micro-
43. Skirrow, M. B., and M. J. Blaser. 2000. Clinical aspects of Campylobacter
infection, p. 969–988. In I. Nashamkin and M. J. Blaser (ed.), Campylobacter,
2nd ed. ASM Press, Washington, D.C.
44. Smith, C. K., P. Kaiser, L. Rothwell, T. Humphrey, P. A. Barrow, and M. A.
Jones. 2005. Campylobacter jejuni-induced cytokine responses in avian cells.
Infect. Immun. 73:2094–2100.
45. Svensson, M., C. Johansson, and M. J. Wick. 2000. Salmonella enterica
serovar Typhimurium-induced maturation of bone marrow-derived dendritic
cells. Infect. Immun. 68:6311–6320.
46. Thoma-Uszynski, S., S. M. Kiertscher, M. T. Ochoa, D. A. Bouis, M. V.
Norgard, K. Miyake, P. J. Godowski, M. D. Roth, and R. L. Modlin. 2000.
Activation of toll-like receptor 2 on human dendritic cells triggers induction
of IL-12, but not IL-10. J. Immunol. 165:3804–3810.
47. Trinchieri, G. 1998. Immunobiology of interleukin-12. Immunol. Res. 17:
48. van der Velden, A. W., M. Velasquez, and M. N. Starnbach. 2003. Salmonella
rapidly kill dendritic cells via a caspase-1-dependent mechanism. J. Immu-
49. Walker, R. I., M. B. Caldwell, E. C. Lee, P. Guerry, T. J. Trust, and G. M.
Ruiz-Palacios. 1986. Pathophysiology of Campylobacter enteritis. Microbiol.
50. Wallis, M. R. 1994. The pathogenesis of Campylobacter jejuni. Br. J. Biomed.
51. Wassenaar, T. M., M. Engelskirchen, S. Park, and A. Lastovica. 1997.
Differential uptake and killing potential of Campylobacter jejuni by human
peripheral monocytes/macrophages. Med. Microbiol. Immunol. (Berlin) 186:
52. Westphal, O., and K. Jann. 1965. Methods in carbohydrate chemistry, vol. 5,
p. 83. Academic Press, New York, N.Y.
53. Wick, M. J. 2002. The role of dendritic cells during Salmonella infection.
Curr. Opin. Immunol. 14:437–443.
54. Wuyts, A., P. Proost, J. P. Lenaerts, A. Ben-Baruch, J. Van Damme, and
J. M. Wang. 1998. Differential usage of the CXC chemokine receptors 1 and
2 by interleukin-8, granulocyte chemotactic protein-2 and epithelial-cell-
derived neutrophil attractant-78. Eur. J. Biochem. 255:67–73.
55. Yao, R., D. H. Burr, and P. Guerry. 1997. CheY-mediated modulation of
Campylobacter jejuni virulence. Mol. Microbiol. 23:1021–1031.
56. Yrlid, U., M. Svensson, C. Johansson, and M. J. Wick. 2000. Salmonella
infection of bone marrow-derived macrophages and dendritic cells: influence
on antigen presentation and initiating an immune response. FEMS Immu-
nol. Med. Microbiol. 27:313–320.
Editor: J. T. Barbieri
VOL. 74, 2006 C. JEJUNI INDUCES DC MATURATION AND CYTOKINE RELEASE 2705