By
From the * Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin;
and Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, 72076 Tübingen, Germany
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-B and the transcriptional activation of inflammatory cytokine
genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent
of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal
infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-
B DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or
CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-
B-specific antibodies, we identified a NF-
B p50/p65 heterodimer. The NF-
B
complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor
and interluekin (IL)-1
occurred at later times and therefore
did not account for NF-
B activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-
B plays a crucial role for the transcriptional activation of
the IL-6 promoter upon Ngo infection. Inactivation of NF-
B conferred by the protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but
not all, studied cyctokine genes. Activation of NF-
B and cytokine mRNA upregulation also
occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.
The activation of transcription factors and the production of immunomodulatory cytokines is an essential
part of the host response to the infection with pathogenic
organisms. In activated T cells, monocytes, and macrophages, genes such as those encoding GM-CSF, the inflammatory cytokines IFN- Cytokines were initially thought to be produced solely
by cells of the immune system; however, it is now evident
that many nonimmune cells, including epithelial cells, produce cytokines. Neisseria gonorrhoeae (Ngo), the etiologic
agent of gonorrhea, can adhere to and penetrate mucosal
epithelial cells during the course of an infection and provoke a strong inflammatory response. Although information about the genetic basis and mechanism of cellular invasion is accumulating, little is known about the cell signaling and its consequences which occur after bacterium-epithelial cell interactions. Analysis of the activated
kinases and transcription factors as well as cytokine gene
upregulation in eukaryotic cells during the infection process may provide first insight into these mechanisms.
A variety of extracellular factors (mitogens, growth factors, bacterial surface components, etc.) initiate the execution of a complex cellular signaling by binding specific
transmembrane receptors onto the eukaryotic cell membrane. The intracellular signaling pathways are complex networks of biochemical reactions that ultimately culminate in
specific patterns of nuclear gene expression mediated by
transcription factors. Preexisting transcription factors that
are involved in immediate early cellular responses can be
posttranslationally activated by a variety of mechanisms. Phosphorylation is the most common modification used to
activate or repress transcription factors (4). Accordingly, extracellular signals that trigger transcription factor activity may
affect transcription factor localization, DNA binding, and
the interaction with the basal transcription machinery. In the
case of the NF- To understand cellular signaling alterations after pathogen infection in epithelial cells better, we examined the altered expression of cytokines and the activation of transcription factors. Here we demonstrate that Ngo induces
inflammatory cytokines and chemokines (TNF- Human Cell Culture and Infection.
Epithelial cells (HeLa, ME180,
HaCaT) were grown in RPMI 1640 (Life Technologies, Eggenheim, Germany) supplemented with 4 mM glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 10% FCS in a humidified 5% CO2 atmosphere. Epithelial cells were seeded in tissue
culture plates for 48 h before infection. 24 h before infection, the
medium was replaced by RPMI 1640 medium supplemented
with 10% FCS and 4 mM glutamine. 2 h before infection, the
medium was again replaced by RPMI 1640 medium supplemented with 0.1% FCS and 4 mM glutamine. The epithelial cells
were infected with Ngo given in MOI in a range of 5-100 for different periods of time. For infection experiments, bacteria were
centrifuged for 5 min at 500 g onto the epithelial cell monolayer.
In the experiments using 2 mM cytochalasin D (Sigma Chemical
Co., St. Louis, MO) or 30 mM TPCK (Sigma Chemical Co.),
the cells were preincubated for 30 min before the bacteria were
added. Stimulation of the cells with 10 ng/ml TNF- Bacteria.
Three different Ngo strains were used for infection
of human epithelial cell lines. The nonpiliated, but invasive Opa+
strain N242 (VP1; P.IA; P RNA Isolation and Reverse Transcriptase PCR.
Total RNA was
isolated using Trizol reagent (Life Tech) as recommended by the
manufacturer's instructions. Total RNA (1 µg) was reverse transcribed into single-stranded cDNA with Superscript IIRT (Life
Tech) and oligo(dt) primers. Amplification of cytokine DNA was
carried out by PCR with the primers indicated in Table 1. We
performed duplex reverse transcriptase (RT)-PCR using Table 1.
Oligonucleotide Primers and PCR Product Sizes for 10 Cytokine cDNAs
, TNF-
, IL-1 and -6, the receptor for IL-2
chain, and the monocyte chemotactic protein (MCP)1-1/JE are highly induced as a result of the regulatory function of the immediate early response factor nuclear factor (NF)-
B (1). Several other transcription factors
have been identified that might play a role in regulating
immune response genes. These include activator protein 1 (AP-1), which is involved in the induction of a variety of
target genes in response to stimulation of cell surface receptors (2) and NF-IL-6 (3).
B/rel family of transcription factors, serine
and tyrosine phosphorylation induce or inhibit a proteolytic degradation of I
B
(5). Nuclear factor
B belongs to a
multigene family of transcription factors that constitute homo-
or heterodimeric proteins with a conserved DNA binding
or dimerization domain (9). In its inactive, cytosolic form,
NF-
B consists of a dimer of DNA-binding subunits bound to an inhibitor, I
B. Dissociation from the inhibitor is triggered by phosphorylation and mostly by degradation of
I
B
(8, 10, 11), and resultant transcription of target genes
can occur in response to a number of agents in a broad
spectrum of cell lines (12). AP-1 transcription factors are a
ubiquitous class of gene regulatory factors that bind specifically to sequences related to the pseudopalindromic AP-1
consensus site (TGA C/G TCA). AP-1 proteins either form
Jun-Jun homodimers comprising the members of the Jun
family (c-Jun, JunD, and JunB) or Fos-Jun heterodimers derived from the various Fos family members (13).
, TGF-
,
GM-CSF, IL-1
, IL-1
, IL-6, IL-8, IL-12, and MCP-1)
in various epithelial cells (HeLa, ME180, HaCaT). Before upregulation of cytokine messenger RNA (mRNA), we
observed activation of the immediate early transcription factor NF-
B. AP-1 was also activated in Ngo-infected cells,
but later in the infection process. Transactivation activity of
NF-
B is induced by invasive or by merely adherent bacteria, even at a multiplicity of infection (MOI) of 5. Deletion
constructs of the IL-6 promoter transfected in HeLa cells
confirmed the importance of the NF-
B enhancer element for the activation of the IL-6 gene in Ngo infected epithelial cells. Moreover, inactivation of NF-
B by the serine
protease inhibitor N-tosyl-L-phenylalanine-chloromethyl
ketone (TPCK), which suppresses the induction of several
cytokine genes, suggests that NF-
B expression is sufficient
for the transcriptional activation of cytokine genes in response to Ngo infection. The activation of NF-
B and
cytokines also occurred in Ngo-infected and cytochalasin D-treated cells, indicating that the cellular signaling is independent of the penetration step of Ngo bacteria.
(Promega,
Heidelberg, Germany) or 40 nM PMA (phorbol 12-myristate 13-acetate; Sigma Chemical Co.) was performed at the indicated
time points.
; Opa27, Opa27.5, Opa28, Opa29, Opa30;
lipopolysaccharide type L1) has been described (14). The adherent, but noninvasive P+ strain N138 (P+ PilEF3) and the P
Opa
control strain N300 (PilEB1; opaC30::cat, pTH7) are derivatives of
strain MS11 (15, 16). Gonococci were routinely grown on phosphate agar as described previously (17). Strain VP1 was additionally grown in 10 ml of RPMI 1640 containing 25 mM Hepes (pH 7.2) for 2 h at 37°C before infection.
-actin primers as an internal control in each reaction and a subsaturating number of cycles allowed a semiquantitative analysis within the infection kinetics. RT-PCR protocols can be obtained upon request. PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis.
Factor
Sequence
Product size
bp
-actin
Sense
5
-GGCACCACACCTTCTACAATGAG-3
838
Antisense
5
-CGTCATACTCCTGCTTGCTGATC-3
IL-1
Sense
5
-GTCTCTGAATCAGAAATCCTTCTATC-3
421
Antisense
5
-CATGTCAAATTTCACTGCTTCATCC-3
IL-1
Sense
5
-AAACAGATGAAGTGCTCCTTCCAGG-3
391
Antisense
5
-TGGAGAACACCACTTGTTGCTCCA-3
IL-6
Sense
5
-ATGAACTCCTCCTCCACAAGCGC-3
627
Antisense
5
-GAAGAGCCCTCAGGCTGGACTG-3
IL-8
Sense
5
-AACATGACTTCCAAGCTGGCC-3
303
Antisense
5
-TTATGAATTCTCAGCCCTCTTC-3
IL-12
Sense
5
-AGGTCAAGGCTATGGTGAGC-3
189
Antisense
5
-TGCCTTCCAGACACTTACGG-3
GM-CSF
Sense
5
-TGGCTGCAGAGCCTGCTGCTC-3
432
Antisense
5
-TCACTCCTGGACTGGCTCCC-3
MCP-1
Sense
5
-ATGAAAGTCTCTGCCGCC-3
303
Antisense
5
-TCAAGTCTTCGGAGTTTGG-3
RANTES
Sense
5
-ATGAAGGTCTCCGCGGCA-3
280
Antisense
5
-CTAGCTCATCTCCAAAGAG-3
TGF-
Sense
5
-GCCCTGGACACCAACTATTGC-3
335
Antisense
5
-GCTGCACTTGCAGGAGCGCAC-3
TNF-
Sense
5
-CAGAGGGAAGAGTTCCCCAG-3
325
Antisense
5
-CCTTGGTCTGGTAGGAGACG-3
ELISA.
Cytokines were assayed in the supernatants of Ngo-infected cells by ELISAs. IL-8 and TNF- ELISA were performed as described by the manufacturer's instructions (R&D
Sys., Minneapolis, MN; and Genzyme Corp., Cambridge, MA).
GM-CSF ELISA used mouse anti-human GM-CSF as capturing
antibody (Genzyme Corp.), rabbit anti-human GM-CSF (Genzyme Corp.) as a secondary antibody, and horseradish peroxidase-labeled goat anti-rabbit IgG (Sigma Chemical Co.) was used
as a tracer antibody.
Preparation of Nuclear Protein Extracts. At the indicated time points after infection, cytoplasmic and nuclear extracts were prepared by using a nonionic detergent method. In brief, the cells were washed and resuspended in buffer A (18) and 0.15% NP-40 was added. The cells were left on ice for 10 min and subsequently centrifuged at 1,000 g for 10 min. The supernatant was used as cytoplasmic extract and the pellet was treated with buffer C (18) for 10 min to yield the nuclear extract. The disruption of the cytosolic membrane appeared to be more efficient than in the Dignam et al. (18) protocol, without affecting the nuclear envelope (10).
Electrophoretic Mobility Shift Assays.
Gel retardation assays for
the detection of NF-B were performed either with an H-2K or
an Ig
oligo probe as described previously (19). The oligonucleotides containing the NF-
B recognition site were labeled using
the large fragment DNA polymerase (Klenow) in the presence of
[32P]desoxy-ATP. The DNA-binding reactions were performed
with 20 µl binding buffer (2 µg poly (dI-dC), 1 µg BSA, 5 mM
dithiothreitol, 20 mM Hepes, pH 8.4, 60 mM KCl, and 10%
glycerol) for 20 min at 30°C. For competition experiments, cold
oligonucleotides were used. Supershifts were performed with antibodies as described previously (10). AP-1 DNA-binding activity
was analyzed using oligonucleotides containing the AP-1-binding
site: 5
-GATCTTCTAGACCGGATGAGTCATAGCTTG-3
; 5
-CAAGCTATGACTCATCCGGTCTAGAAGATC-3
. The
AP-1 DNA-binding oligonucleotide was labeled using T4 kinase
(Boehringer Mannheim GmbH, Mannheim, Germany) in the presence of
[32P]desoxy-ATP. DNA-binding reactions were performed using a binding buffer containing 2 µg poly (dI-dC), 1 µg
BSA, 10 mM Tris, pH 7.5, 10 mM MgCl2, 100 mM KCl, 1 mM
EDTA, 1 mM dithiothreitol, and 10% glycerol. Other used oligonucleotides containing DNA-binding sites: cAMP-responsive
element (CRE): 5
-CGAGCCCCATTGACGTCAATGGGGTCGAC-3
, 5
-GTCGCCCCATTGACGTCAATGGGGCTCG-3
; CAAT/enhancer binding protein (C/EBP): 5
-TGCAGATTGCGCAATCTGCA-3
, 5
-TGCAGATTGCGCAATCTGCA-3
; octamer H2B: 5
-GATCCCAACTCTTCACCTTATTTGCATAAGCGATTCTATAG-3
, 5
-GATCCTATAGAATCGCTTATGCAAATAAGGTGAAGAGTTGG-3
. For
the CRE and C/EBP oligonucleotides, the same buffer conditions as indicated for AP-1 were used, whereas for the octamer-binding site NF-
B, shift conditions were used. The reaction
products were analyzed by electrophoresis in a 5% polyacrylamide
gel using 12.5 mM Tris, 12.5 mM Boric acid, and 0.25 mM
EDTA, pH 8.3. The gels were dried and exposed to Amersham
TM film (Amersham Buchler, Braunschweig, Germany) at
70°C
using an intensifying screen.
Luciferase and Human Growth Hormone Reporter Assays.
HeLa,
HaCat, or ME180 cells at 50-70% confluence were transfected by
cationic liposomes (Promega) with 1 µg of a luciferase expression
plasmid containing four repeats of the NF-B H-2K-binding site
as enhancer element. 24 h after transfection, cells were either infected with Ngo strains, stimulated with PMA or TNF-
, or treated
with LPS. Luciferase assays were performed at the indicated time
points as recommended by the manufacturer's instructions (Promega).
The results were recorded on a
-counter (Beckman Wallac 409;
Beckman, Munich, Germany). The data presented are representative of more than three separate experiments.
To assess changes in cytokine gene expression of human epithelial cells in response to bacterial
infection, ME180, HeLa, and HaCa T cells were challenged to infection with Ngo at a MOI of 5, as described in
Materials and Methods. The assays were performed with
different Ngo strains and cytokine expression was determined at different time points after infection, i.e., 0, 15, 45, and 120 min, by reverse transcription (RT-PCR) of total
RNA prepared from infected cells. The cytokine mRNA
levels were compared with the constitutive -actin mRNA
in the same RT-PCR reaction. The Ngo strains used differed with regard to the infection of epithelial cells. Strain
N242 (Opa+) enters epithelial cells by binding to heparan
sulfate containing surface proteoglycan receptor proteins
(14, 20, and Dehio, C., E. Freissler, C. Lanz, O. Gomez-Duarte, G. David, and T.F. Meyer, manuscript submitted).
Strain N138 (P+ Opa
) expresses pili that also confer adherence to epithelial cells (21), but does not express any
Opa proteins and therefore is not invasive (14). Strain
N300 (P
Opa
) is neither piliated, nor does it express
Opa proteins (16); consequently this strain adheres only
weakly to human cells. Gonococcal infection was routinely
monitored by light microscopy of the bacteria using the
crystal violet staining technique (22).
Each of the three studied epithelial cell lines expressed
inflammatory cytokines in response to Ngo infection. As
shown in Fig. 1 A, infection of ME180 cells with Ngo led
to an increased synthesis of several cytokines, i.e., GM-CSF, TNF-, IL-8, MCP-1, TGF-
, and IL-1
as soon as
15 min after infection. Maximum expression was achieved
2 h after infection. Furthermore, the cytokine IL-6 was induced within 45 min after infection and the cytokines IL-1
and IL-12 showed constitutive mRNA expression with a
slight increase upon infection. The chemokine RANTES
(regulated on activation, normal T cell expressed and secreted) was constitutively expressed without change in infected cells. Both the adherent (P+) and the invasive (Opa+)
gonococci were efficient in cytokine upregulation, whereas
the P
Opa
gonococci did not significantly increase cytokine mRNA levels. LPS, a major constituent of the gram-negative bacterial outer membrane and a potent inducer of
cytokine mRNA upregulation in CD14 positive cells, did
not effect cytokine expression (data not shown). None of
the tested cell lines induced detectable levels of mRNA
coding for IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, intercrine 309 (I-309), or IFN-
(data not shown).
These studies indicate a coordinate upregulation of inflammatory cytokine mRNA levels in epithelial cells upon
infection with Ngo. To demonstrate for some of these cytokines that their increased mRNA levels actually lead to
an increased protein secretion, we analyzed the cytokine
release by ME180 cells in response to bacterial infection. As
shown in Fig. 1 B, infection with the P+ and Opa+ Ngo
strains, respectively, resulted in an increase of GM-CSF, IL-8, and TNF- secretion. The release was weak until 3 h
and increased steadily during the following time. The P
Opa
control strain induced GM-CSF, IL-8, and TNF-
release to some extent at 6 h after infection.
Ngo infection in epithelial cells may also induce alterations in signal transduction pathways that modulate cellular transcription factors. Moreover, the activation of transcription factors and inflammatory cytokine gene expression may be coordinately inducible in Ngo-infected cells. Therefore, we investigated whether Ngo infection induces transcription factors that could be responsible for inflammatory cytokine gene expression.
Subconfluent monolayers of HeLa cells were infected
with Ngo. At different time points after challenge, nuclear
protein extracts were prepared and analyzed for the levels
of cellular transcription factor DNA-binding activity by using a panel of radiolabeled oligonucleotides corresponding
to the DNA-binding sites of five transcription factor families (NF-B, AP-1, CRE, C/EBP, and octamer factors). As
Fig. 2 A shows, when the NF-
B-binding site (H-2K) was
used for the electrophoretic mobility shift assay (EMSA), an
enhanced binding of nuclear proteins was observed in
HeLa cells within 10 min after infection with the P+ strain
(Fig. 2 A, lanes 1 and 2). The DNA-binding activity increased within 90 min, and was already reduced after 180 min (Fig. 2 A, lanes 3-5). To examine the specificity of the
DNA-binding capability induced by adherence of the P+
strain, nonlabeled double-stranded oligonucleotide was added for competition. A decrease in the amount of bound complex was observed as the concentrations of unlabeled NF-
B
consensus sequence increased (Fig. 2 A, lanes 6-9). The
most prominent form of transcription factor NF-
B has
been described as a heterodimer consisting of two proteins,
p50 and p65 (23). Furthermore, members of the NF-
B/rel
family of proteins can form homodimers and heterodimers (24). The nature of the proteins that bind to the
B sequence were characterized using supershift assays. Experiments were performed in which the nuclear extracts were
preincubated with either an anti-p50, anti-p65, or anti-c-Rel
antiserum before addition of the 32P-labeled oligonucleotide. The results shown in Fig. 2 A (lanes 10-15) indicate
that the anti-p50 antibody (0.5 µl and 2 µl antiserum, lanes
10 and 11) and the anti-p65 antibody (0.5 µl and 2 µl antiserum, lanes 12 and 13) led to a significant reduction and to
supershifts of the NF-
B complex. The anti-c-Rel antibody
affected only slightly the NF-
B-DNA complex (0.5 µl and 2 µl antiserum, lanes 14 and 15). Therefore, both p50 and p65
represent the predominant protein species in the
B DNA-binding activity present in P+ strain-infected HeLa cells.
To evaluate whether cellular NF-B activation is at variance in epithelial cells infected with adherent versus invasive Ngo strains, we compared NF-
B DNA-binding activity in HeLa cells infected with either the P+ or Opa+ strain.
At a MOI of 100, the noninvasive P+ strain induced stronger and earlier activation of NF-
B than the Opa+ strain,
whereas no NF-
B activation was observed in cells infected with the P
Opa
control strain or LPS-treated cells (Fig. 2 B).
The activation of NF-
B in response to Ngo infection was
delayed compared to the NF-
B activation in response to
TNF-
, but showed a similar potential to induce DNA-binding activity. The activation of the immediate early transcription factor NF-
B in Ngo-infected epithelial cells was also inducible at a MOI of 5. This indicates highly specific Ngo-induced signaling leading to downstream activation of NF-
B.
Activation of AP-1 in response to Ngo infection was
studied using an AP-1 consensus DNA-binding oligonucleotide. Protein-DNA complexes were identified in HeLa
cells infected with either the P+ or the Opa+ strain in
EMSA using an oligonucleotide containing the AP-1-binding site, indicating that members of the c-jun/c-fos family were
activated (Fig. 2 C). AP-1 DNA-binding activity was observed 90 min after infection and was as strong as AP-1 activation in response to PMA, which induces AP-1 within 10 min. No AP-1 activation was observed in N300 (P Opa
)
-infected or LPS-treated cells. In contrast to the experiments performed to study NF-
B activation in response to
Ngo infection, AP-1 activation was only observed reproducibly in epithelial cells, which were infected in the presence of FCS. The removal of serum of epithelial cells induced AP-1 activation leading to a constitutive high AP-1
DNA-binding activity at the time of infection, which became enhanced only marginally during Ngo infection.
The DNA-binding capability of other transcription factors investigated with oligonucleotides containing the consensus recognition sequence for other basic leucine zipper factors, the C/EBP and the palindromic CRE, as well as the octamer-binding site, were not enhanced by Ngo infection (Fig. 2 D).
Ngo-Epithelial Cell Interaction Induces Activation of NF-The highly invasive Opa+ Ngo strain was used to determine whether cellular invasion by the bacteria was a prerequisite for the activation of inflammatory cytokines and transcription factors. Previously, we demonstrated the apparent role of the actin cytoskeleton in the gonococcal entry process by the inhibition of gonoccocal uptake by cytochalasin D (14, 17). Since the recruitment of F-actin by Ngo is not affected by cytochalasin D treatment, we assume the integrity of the actin microfilaments is required to complete the bacterial entry process.
To study whether signaling in HeLa cells infected with
Opa+ Ngo is dependent on the bacterial penetration step,
we infected the eukaryotic cells either in the presence or
absence of 2 mM cytochalasin D. As shown in Fig. 3, despite of the treatment with cytochalasin D, the Opa+ Ngo
were still able to efficiently activate the transcription factor
NF-B. The observation that inhibition of epithelial cell invasion by cytochalasin D treatment does not cause alterations in the downstream signaling suggests that adherence,
rather than cellular invasion, is responsible for the induction of the signal. This assumption was supported by cytokine expression data obtained by measuring the release of
TNF-
, GM-CSF, and IL-8 in VP1-infected and cytochalasin-treated HeLa cells. Herein, Opa+ Ngo-infected HeLa
cells induced a cytokine release that was at similar levels independent of the presence or absence of cytochalasin D
(data not shown).
Ngo Infection Induces Transactivation at the NF-
To quantify the NF-B activation in epithelial
cells elicited by Ngo infection, HeLa cells were transfected
with a plasmid (pGL2-Luc/H-2K) containing four tandem
repeats of the H-2K NF-
B DNA-binding sequence
linked to a luciferase gene. As shown in Fig. 4 A, epithelial
HeLa cells transfected with this promoter construct exhibited a substantial capacity to express luciferase activity in response to different stimuli. Cells infected at a MOI of 50 with either the P+ or the Opa+ strain, and TNF-
-treated
cells showed a rapid increase in luciferase activity (~three- to
sixfold) within 15 min. In cells infected with the P+ strain,
luciferase activity steadily increased during the first 90 min
showing a ~16-fold activation as compared to nontreated cells, whereas cells infected with the invasive (Opa+) strain,
and TNF-
-treated cells have their maximal luciferase activity (9- and 11-fold, respectively) 45 min after infection. In all cases, NF-
B transactivation decreased rapidly from
the peak activity to the near basal level within 180 min.
Control Ngo (P
Opa
) -infected cells or LPS-treated HeLa
cells showed no enhanced NF-
B transactivation compared
to the nontreated control cells.
The inducibility and strength of the NF-B transactivation is influenced by the MOI as shown in Fig. 4 B. Infection of HeLa cells with P+ Ngo at a MOI of 5 or 50 induced an approximately sixfold higher luciferase activity
compared to noninfected cells at 45 min after infection. In
contrast to the infection of cells at MOI 50, the luciferase
activity in cells infected with MOI 5 declines rapidly.
The level of NF-B transactivation depends on the epithelial cell type (Fig. 4 C). The highest level of transactivation in response to P+ Ngo infection was observed in HeLa
cells (16-fold within 90 min after infection), whereas HaCaT
and ME180 cells revealed an approximately fourfold NF-
B
transactivation.
As shown in Fig. 1 A, IL-6 mRNA is upregulated in
response to Ngo infection. Therefore, the following experiment was aimed to investigate the molecular mechanism
mediating this effect. To this end, deleted forms of IL-6
promoter fragments linked to the hGH gene as a reporter
gene were used (Fig. 5 A), and plasmids were transiently
transfected into HeLa cells. Promoter activity was assayed
after Ngo infection by analysing the activity of the hGH in
cell-free culture supernatants. Infection experiments were performed for 3 h in the presence of 10% FCS. Transcriptional induction by infection with P+ Ngo was observed in
all promoter constructs with the exception of the promoter
construct deleted upstream of position 49, containing no
enhancer elements. As much as approximately threefold
hGH activity was observed from the longest promoter construct deleted upstream of position
524 containing the
NF-
B, NF-IL-6, and AP-1 enhancer elements (Fig. 5 A).
With this construct, the exchange of the medium before
the infection already led to high hGH levels (Fig. 5 B).
Transfection of the promoter construct upstream of position
219 lacking the binding site for the transcription factor AP-1 (position
283 to
277) caused a drastic reduction of the residual hGH release, but did not affect the
inducibility of hGH release by Ngo infection. Although the
NF-IL-6-binding site at position
158 to
145 has been
demonstrated to be involved in transcriptional activation of
the IL-6 promoter (25), we observed no activation of NF-
IL-6 at the C/EBP DNA-binding site in response to Ngo
infection by EMSA (Fig. 2 D). Thus, NF-IL-6 does not
appear to contribute in IL-6 promoter activation. These data
rather suggest that NF-
B plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection.
Ngo Infection Induces Cytokine mRNA Upregulation Is Blocked by NF-
From the experiments described above, we suggest that the activation of the transcription factor NF-B represents a critical event in Ngo infection of HeLa cells. To study the importance of NF-
B
in the downstream signaling after Ngo infection, we asked whether the blockage of NF-
B activation by the addition
of the serine protease inhibitor TPCK could cause a block
of cytokine mRNA upregulation. As shown in Fig. 6 A,
TPCK effectively blocked I
B
degradation and NF-
B
activation in response to P+ Ngo infection as determined in
the gel retardation assay. The TPCK inhibitor does not
generally or nonspecifically affect DNA-binding proteins
since, for example, Oct-1 DNA-binding activity remains unaffected (data not shown). To determine if cytokine
mRNA upregulation is affected by Ngo infection of TPCK
pretreated cells, we investigated a panel of cytokine genes
as shown in Fig. 1 A. TPCK pretreatment of HeLa cells led
to a strong inhibition of GM-CSF, TNF-
, IL-8, IL-6, and
MCP-1 cytokines mRNAs synthesis upon infection with P+ Ngo (Fig. 6 B). Marginal or no blockage of mRNA synthesis was observed for the cytokines IL-1
, IL-1
, and
TGF-
. Thus, in most of the cases studied, the activation
of the immediate early factor NF-
B is sufficient for the
transcriptional activation of cytokine genes in response to
infection with Ngo.
Since the mucosal epithelial surface forms the first barrier that impedes the penetration of microorganisms, we used this cell type to study the activation of cytokine expression as a response to an infection with Ngo. Cytokines are important modulators of the antigen-specific immune response and thus influence the immune surveillance. If cytokines are induced upon the initial contact of a pathogen with mucosal surfaces, this response could be crucial to the outcome of an infection. Our current investigation supports this notion and reveals that Ngo induces the upregulation of a variety of so-called proinflammatory cytokines. Infection with either adherent P+ Ngo or invasive Opa+ Ngo leads to a rapid, direct, and specific activation of a panel of cytokine genes. Our data indicate that physical invasion of epithelial cells by Ngo is not a prerequisite of cytokine upregulation. Thus, it appears that the bacterium eukaryotic cell contact is already sufficient to induce an efficient downstream signaling.
The upregulation of cytokine mRNA and the release of
cytokines were observed in several different human epithelial cell lines. The Ngo-induced cytokines TNF-, IL-6, and
IL-1 have been implicated in the defense of bacterial infections and promote bactericidal activity of leukocytes. In addition, the three cytokines can activate T and B lymphocytes. Their primary function may therefore be host protective
(26), although adverse effects towards the host cannot be
ruled out. The other cytokines induced by Ngo infection
have a major inflammatory activity. The cytokine GM-CSF
induces granulocyte/macrophage populations and TGF-
represents a neutrophil chemoattractant. Moreover, TGF-
also exerts activities that oppose or downregulate inflammatory processes. The chemokine IL-8 induces leukocyte
chemotaxis and the chemokine MCP-1 attracts monocytes/macrophages. As epithelial cells represent the entry
sites for bacterial infections, the activation of these chemokines
functions as an early warning system at a time when bacterial products are unavailable to the stimulation of circulating leukocytes (26). The array of proinflammatory cytokines produced by epithelial cells overlaps, but also exhibits several important differences from that produced by cells of
the monocyte/macrophage lineage. Thus, no high levels of
IL-12 p40 or IL-1 were expressed by epithelial cells (27,
28). Furthermore, the array of cytokines expressed by epithelial cells markedly differs from that characteristic of T
cells, mast cells, eosinophils, and NK cells (29). Thus,
none of the Ngo-infected epithelial cells expressed mRNA
for IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, I-309, and IFN-
.
Taken together, our findings support the notion that epithelial cells are an integral component of the host's nonspecific immune system. This concept is consistent with other reports. For example, E. coli induces IL-1
, IL-1
, IL-6,
and IL-8, but not TNF-
in epithelial cells (32, 33). Invasive Salmonella, Yersinia, Shigella, and Listeria species have
been shown to activate a wide range of cytokines like
MCP-1, GM-CSF, IL-8, and TNF-
(34, 35). Herein, the
bacterial attachment seems to be the signal enhancing the
epithelial cell cytokine responses (33, 36). LPS effectively
induces cytokine production from macrophages, but only
poorly induces epithelial cytokine responses. The poor LPS
response may be explained in part by lack of CD14 on epithelial cells (37).
The rapid production of proinflammatory cytokines by
epithelial cells involves the activation of immediate early
transcriptional activators. Cellular transcription factors are
participants in the host response to infection. The promoters of genes important in the immune response like TNF-,
IL-6, IL-8, and MCP-1 contain binding domains for a
number of transcription factors, including NF-
B. As confirmed by many other studies, the immediate early transcription factor NF-
B plays a critical role in host immune response to bacterial and viral pathogens (9). Gel retardation assays using nuclear extracts from Ngo-infected epithelial cells and a
B consensus motif resulted in activation of
NF-
B-rel complexes as compared with nuclear extracts
from uninfected cells. Supershift experiments with antibodies against p50 and p65 led to upshifts or the disappearance
of the NF-
B complex, suggesting that the protein-DNA
complex is composed of the p50 and p65 proteins. The NF-
B activation in epithelial cells occurred within 10 min
after infection with P+ Ngo at an MOI of 100 and the induction of cytokine mRNAs were detected after 15 min of
infection. High specificity for NF-
B activation was evident from Ngo infection experiments using a MOI of 5, which also showed strong NF-
B DNA binding in the
EMSA, even with a delay in time. Similarly, transient
transfection assays revealed that the NF-
B site is strongly
activated in response to Ngo infection (Fig. 4). In contrast
to NF-
B, AP-1 was induced at an MOI of 100 within 90 min after infection. Further, transcription factors like C/EBP,
CRE, or octamer DNA-binding factors tested for their activation in Ngo-infected epithelial cells were either not activated or maintained their activity.
Experiments with cultured epithelial cells suggest that
gonococcal entry into epithelial cells involves both the polymerization of actin microfilaments (14, 17) and microtubuli (38, and Dehio, C., E. Freissler, C. Lanz, O. Gomez-Duarte, G. David, and T.F. Meyer, manuscript submitted).
Since we speculated that NF-B activation and cytokine activation might already be triggered by adherent Ngo through
as yet undefined surface proteins, we studied the cytokine
activation in the presence of the microfilament disrupting
agent cytochalasin D. In our experiments, we observed that
the inhibition of the internalization of invasive Ngo by cytochalasin D affected neither NF-
B DNA binding (Fig. 3)
nor cytokine release (data not shown) supporting the notion that Ngo adhesion to the surface of epithelial cells triggers signals leading to NF-
B activation. An invasion independent activation of NF-
B was described for Listeria
monocytogenes on macrophages (39), whereas in the case of
Shigella flexneri invasion was discussed as a prerequisite for
NF-
B activation (40).
The transient transfection assay using deletion mutants of
the IL-6 promoter revealed that the NF-B site is primarily
responsible for Ngo-dependent induction of IL-6 (Fig. 5).
This is in line with other studies that have shown that the
NF-
B-binding site is crucial for the activation of the IL-6
promoter (41). Both the induction of the IL-6 mRNA and
the activation of the transcription factor NF-
B occur rapidly, indicating that Ngo infection causes posttranslational
modifications of preexisting NF-
B. AP-1, as well as the
NF-IL-6 transcription factor, have previously been shown
to be involved in the transcriptional activation of the IL-6
gene by compounds such as PMA (42). Some evidence,
however, indicates that AP-1 and NF-IL-6 do not participate in the immediate Ngo-induced upregulation of IL-6
transcripts. Transcriptional activation of the IL-6 promoter
requires an intact NF-
B site, whereas deletion of the AP-1
enhancer element does not abolish inducibility by Ngo
infection. Further, EMSA data clearly show that AP-1
DNA-binding activity occurs later than IL-6 mRNA upregulation, and DNA-binding activity of NF-IL-6 at the
C/EBP-binding site in response to Ngo infection was not
observed.
To test if NF-B could sufficiently contribute to cytokine mRNA upregulation, we inhibited the degradation of
the NF-
B inhibitor I
B
, which usually undergoes complete proteolytic degradation after NF-
B stimulation (24).
Inhibition of I
B
degradation and NF-
B activation was
achieved using the cell-permeable serine protease inhibitor
TPCK. Under these conditions, concomitant infection of
HeLa cells with P+ Ngo inhibited the upregulation of cytokine mRNAs of TNF-
, GM-CSF, IL-6, IL-8, and
MCP-1 (Fig. 6). Thus, the immediate early transcription factor NF-
B has a major role in the transactivation of
these cytokine genes in response to Ngo infection. In contrast, the cytokine gene promoters of IL-1
, IL-1
, and
TGF-
, which exert either weak or no NF-
B enhancer
elements, still showed upregulation of their mRNAs in response to Ngo infection. Interestingly, the elimination of
the transactivation activity of NF-
B is used as an immune
evasion strategy by the African swine fever virus. This virus
produces an inhibitor of NF-
B, A238L, which shows homology to I
B
. A238L interacts with NF-
B to prevent
transcription and causes an almost complete shutdown of
proinflammatory cytokines (43).
Since both the adherent P+ Ngo and the invasive Opa+
Ngo strongly induce NF-B, AP-1, and proinflammatory
cytokines, we assume complex bacterial stimuli are responsible for triggering the multiple signals in human cells. Experiments are now in progress to determine which signaling
pathways could account for the induction of the transcription factors NF-
B and AP-1 in Ngo-infected epithelial cells.
Address correspondence to Dr. Michael Naumann, Max-Planck-Institut f ür Infektionsbiologie, Abt. Molekulare Biologie, Monbijoustr. 2, 10117 Berlin, Germany. Phone: 49-30-28026317; FAX: 49-30-28026611; E-mail: naumann{at}mpiib-berlin.mpg.de
Received for publication 7 April 1997 and in revised form 6 May 1997.
1Abbreviations used in this paper: AP-1, activator protein 1; C/EBP, CAAT/ enhancer-binding protein; CRE, cAMP-responsive element; EMSA, electrophoretic mobility shift assay; hGH, human growth hormone; I-309, intercrine 309; MCP, monocyte chemotactic protein; MOI, multiplicity of infection; mRNA, messenger RNA; NF, nuclear factor; Ngo, Neisseria gonorrhoeae; RT-PCR, reverse transcriptase PCR; TPCK, N-tosyl-L-phenylalanine-chloromethyl ketone.We thank M.A. Brach for providing the IL-6 promoter constructs.
This work was supported in part by the Fonds der Chemischen Industrie.
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