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Infection and Immunity, November 1999, p. 6056-6066, Vol. 67, No. 11
Unité de Pathogénie Microbienne
Moléculaire/Unité INSERM 3891 and
Unité de Pharmacologie Cellulaire/Unité INSERM
485,2 Institut Pasteur, 75724 Paris Cedex 15, France; Department of Pathology, Kumamoto University Medical
School, Kumamoto 860, Japan3; and
Department of Microbiology, Skirball Institute, New York
University Medical Center, New York, New York
100164
Received 24 March 1999/Returned for modification 11 May
1999/Accepted 5 August 1999
Infection by the enteric bacterial pathogen Shigella
results in intense mucosal inflammation and destruction of the colonic and rectal epithelium in infected humans. Initial bacterial
translocation occurs through the follicle-associated epithelium.
Previous experiments suggest that interleukin-1 (IL-1) is crucial to
trigger inflammation, particularly in the follicular zones. During the
first 4 hours of infection in a rabbit ligated-loop model of intestinal
invasion, there are two salient characteristics: (i) a high
concentration of IL-1 Shigellosis is an acute inflammatory
disease of the colonic and rectal mucosae in humans. In its major form,
mucosal tissue destruction generated by acute inflammation causes the
dysenteric symptoms characterized by bloody, mucopurulent stools.
Shigella, the causative agent, invades the colonic and
rectal mucosae, particularly epithelial cells (37).
Shigella is a gram-negative bacterium belonging to the
family Enterobacteriaceae. Acute inflammation following an
infection, essentially by recruitment and activation of cells of the
innate immune system, such as monocytes and polymorphonuclear leukocytes (PMN), is the cost of rapid eradication of the invading pathogen. The acute inflammation generated by Shigella is
initially rich in PMN. In shigellosis, inflammation has the paradoxical effect of causing severe damage to the mucosa and allowing further bacterial invasion before the inflammatory response controls the infection (47, 48, 55). Full-blown shigellosis is
characterized by severe diffuse intestinal inflammation extending
beyond areas of mucosal invasion (27) and by complications
reflecting uncontrolled local and systemic inflammation, such as toxic
megacolon, colonic perforations, pseudoleukemoid syndrome, and the
hemolytic-uremic syndrome (27).
A balance must be maintained in intestinal tissues between inductive
and inhibitory signals of inflammation in order to provide protection
from the bacterial products released by the commensal flora and to
allow proper conditions for healing and regeneration in case of
aggression by a noxious agent or a pathogen. This is illustrated by the
occurrence of an acute enterocolitis in interleukin-10 (IL-10), IL-2,
and T-cell receptor-knockout mice harboring normal intestinal flora but
its absence in their germ-free counterparts (35). Invasive
bacterial pathogens such as Shigella, which cause acute
intestinal inflammation, have probably evolved strategies to
up-regulate proinflammatory signals and down-regulate anti-inflammatory signals. The gut inflammation observed in shigellosis resembles other
infectious and noninfectious colites, such as idiopathic inflammatory
bowel diseases (IBD). Moreover, the histopathological lesions of
shigellosis are similar to those of acute-stage ulcerative colitis.
Initial translocation of Shigella through the epithelial
barrier occurs via M cells of the follicle-associated epithelium (FAE)
(53, 56, 59). This makes colonic and rectal lymphoid solitary nodules and their associated dome the major site of initial inflammation, which may then extend, due to the capacity of
Shigella to enter cells at their basolateral poles
(46) and to spread from cell to cell (8).
Upon contact with target cells, Shigella secretes four
invasion plasmid antigens (Ipa proteins) via a type III secretory
apparatus called Mxi-Spa (42-44). In the presence of
epithelial cells, Ipa proteins induce major cytoskeletal rearrangements
which mediate bacterial entry via macropinocytosis (1). The
bacteria then spread from cell to cell via an actin-dependent process
involving IcsA, a protein polarly exposed at the bacterial surface,
which nucleates and amplifies actin polymerization (8, 23,
39). In the presence of macrophages, IpaB causes rapid apoptotic
death of these cells both in vitro (12, 64, 67) and in vivo
(33, 66).
When macrophages are preactivated with lipopolysaccharide (LPS) and
subsequently infected by invasive Shigella flexneri, they release large quantities of mature IL-1 We have previously shown that by initiating inflammation, IL-1 accounts
for a rupture of the epithelial barrier that enhances bacterial
invasion (47, 48) and initiates a cascade leading to tissue
destruction. Treatment of infected rabbits with IL-1 receptor
antagonist (IL-1ra) can significantly reverse both effects (55). Here we show that in the course of experimental
shigellosis in the rabbit ligated-loop infection model, the balance
between IL-1ra and IL-1 expression in infected follicular structures is severely impaired at the early stage of bacterial invasion, possibly due to macrophage apoptosis. Insufficient expression of IL-1ra may
largely account for the severity of inflammation.
Bacterial strains and culture conditions.
M90T is a
wild-type S. flexneri serotype 5 strain (54).
BS15 is a noninvasive derivative of M90T which was cured of the 220-kb
plasmid pWR100 and transformed with the recombinant plasmid pRKB15
(66) encoding AFR1, an adherence pilus of the
rabbit-specific Escherichia coli strain RDEC-1
(13). Prior to experimental infection of rabbits, the
bacteria were streaked on Congo red agar. Congo red-positive colonies
were resuspended in tryptic soy broth (Diagnostics Pasteur, Marnes la
Coquette, France), plated on tryptic soy agar plates, and cultured
overnight to confluency at 37°C. The bacteria were harvested in
sterile 0.9% NaCl, and their concentration was adjusted to
1010 bacteria per ml.
Experimental Shigella infection of rabbits.
A
total of 24 specific-pathogen-free male New Zealand White rabbits,
weighing between 2.5 and 3 kg (CEGAV S.S.C., Les Hautes Noës,
France), were used in this study. Twelve animals were infected with
M90T, and 12 animals were infected with BS15. In each group, four
animals were sacrificed 2, 4, or 8 h postinfection. The rabbits received general anesthesia combining acepromazine (250 mg/kg of body
weight) (Calmivet, Magny-Vernois, France) intravenously (i.v.) and
ketamine (20 mg/kg) (Rhône Mérieux, Lyon, France) i.v.
After a laparotomy, ligated loops 5 cm long were made, each containing
a Peyer's patch. Depending on the number of Peyer's patches
available, between five and six loops were made in each rabbit.
Ligations were carefully performed to preserve the afferent and
efferent mesenteric vasculature. Inocula of 5 × 109
bacteria in 0.5 ml were injected in each loop. The abdominal cavity was
then closed. Two, 4, and 8 h postinfection, the rabbits were again
anesthetized with ketamine (20 mg/kg) and laparotomized. The mesenteric
vein draining each infected loop was sampled with a 26-gauge by 1/2-in.
needle mounted on a 1-ml syringe, and 50 to 100 µl of blood was
aspirated and immediately mixed with an anticoagulant and a cocktail of
protease inhibitors (0.05% [wt/vol] sodium azide, 1 mg of
aprotinin/ml, 1 mg of leupeptin/ml, 1 mg of pepstatin A/ml, and 1 mM
AEBSF [all components from Sigma]). Plasma was collected after
centrifugation and conserved at Bacterial counts in tissue samples.
Peyer's patches from
animals infected for 2, 4, or 8 h were punched with a skin biopsy
device, providing a disk 6 mm in diameter. The biopsied tissue samples
were immersed in a gentamicin solution and washed extensively in cold
0.1× PBS. Tissue samples were then ground with an Ultra-Turrax
apparatus and resuspended in a final volume of 1 ml. Dilutions were
spread on tryptic soy agar plates after a 30-min incubation at 37°C.
Colonies were counted, and CFU were calculated for 1 ml of tissue homogenate.
Immunohistochemistry.
Tissues were fixed in 4% formalin,
dehydrated, and embedded in historesin (Leica Instruments, Heidelberg,
Germany). Blocks were sectioned in 5-µm-thick slices. Immunostaining
of the sections was performed with the following antibodies: LPS was
labeled by using a biotinylated mouse monoclonal antibody
(immunoglobulin G3, kappa chain) directed against the S. flexneri serotype 5 somatic antigen (49). IL-1 In situ detection of fragmented DNA.
Terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) was performed as described by Gavrieli et al. (22).
Deparaffinized tissue sections were sequentially treated with acetone
and proteinase K (Boehringer Mannheim, Indianapolis, Ind.). Labeling
was done with the apoptosis detection system fluorescein kit (Promega,
Madison, Wis.). Each field was scanned with a confocal microscope
(Leica) in both channels. The sections were reconstructed with Adobe Photoshop.
Quantification of IL-1 IL-1 Titration of mRNA for IL-1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Increased Interleukin-1 (IL-1) and Imbalance
between IL-1 and IL-1 Receptor Antagonist during Acute Inflammation in
Experimental Shigellosis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and IL-1
, both in infected Peyer's patch
tissue and in the corresponding efferent mesenteric blood, and (ii) a
very low level of expression of IL-1 receptor antagonist (IL-1ra). These may reflect a combination of regulation of expression and secretion of IL-1, IL-1, and IL-1ra by both resident and
recruited phagocytes and the induction of mononuclear phagocyte
apoptosis by Shigella. This low IL-1ra/IL-1 ratio likely
accounts for the rapid, uncontrolled inflammation characteristic of shigellosis.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(65). This
reflects direct activation of ICE (caspase 1) by IpaB in infected
macrophages (12, 31). Shigella is therefore able
to achieve a dual function (63) in a single cell population:
cell killing, which may protect the invasive microorganism against
rapid eradication by phagocytic cells in subepithelial tissues, and
initiation of early inflammation mediated by IL-1.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C. The animals were then
sacrificed by i.v. injection of a 10-ml air bolus. The loops were
quickly dissected and opened, and sections of the Peyer's patches were
sampled with a skin biopsy punch providing disks 8 mm in diameter
(Stiefel, Nanterre, France). The biopsy material was immediately cut
into two equal pieces. One half was frozen in liquid nitrogen and
stored at 80°C for further extraction of mRNA. The other half was
put in 2 ml of cold phosphate-buffered saline (PBS) containing a
cocktail of protease inhibitors (see above) and homogenized in ice for
10 s with an Ultra-Turrax homogenizer (Janke & Kunkel GmbH,
Staufen, Germany). The samples were then centrifuged at
100,000 × g for 1 h at 15°C, and the
supernatants were immediately stored at 80°C for cytokine quantification.
,
IL-1, and IL-1ra were labeled by their respective specific
monoclonal antibodies, which will be identified below. A RAM11-specific
monoclonal antibody (Dako Corporation, Carpinteria, Calif.) was used to
detect the macrophage population expressing this molecule. In order to
label the primary antibody, the LSAB Kit-Peroxidase (Dako Corporation)
was used. Briefly, the technique is based on the streptavidin biotin
reaction. Endogenous peroxidase activity is quenched by first
incubating the samples in 3% H2O2 for 5 min.
The samples are then incubated with the primary antibody, followed by
sequential 10-min incubations with biotinylated antibody and
peroxidase-labeled streptavidin. Amplification is obtained by 30-min
incubation with the ABC peroxidase kit (Vectastain ABC; Vector
Laboratories, Inc., Burlingame, Calif.). Staining is obtained by a
10-min incubation with the freshly prepared substrate chromogen (DAB)
solution (3,3'-diaminobenzidine
tetrahydrochloride-H2O2 [Dako]).
Counterstaining is obtained with Harris hematoxylin (Merck, Rahway,
N.J.). Final mounting is done on Glycerogel (Dako).
and IL-1ra.
Stored plasmas and
supernatants of Peyer's patch homogenates were tested in a sandwich
enzyme-linked immunosorbent assay employing antibodies specific for
rabbit IL-1 and IL-1ra as described previously (41).
Briefly, the wells of microtiter plates were coated with Na carbonate
buffer (pH 9.6) with either a mouse monoclonal immunoglobulin G
directed against IL-1ra or a sheep polyclonal serum directed against
both pro- and mature rabbit IL-1. After being washed with PBS-Tween
20 (0.05%) and saturated with PBS-bovine serum albumin (1%), plaques
were incubated overnight at 4°C with 100 µl of sample. They were
washed again before anti-IL-1 or anti-IL-1ra biotinylated antibodies
were added at a concentration of 5 µg/ml; the reaction was amplified
by 100 µl of the avidin-biotin peroxidase complex (Vectastain; Vector
Laboratories). After being washed, the enzyme-linked immunosorbent
assay reaction was revealed by the addition of 100 µl of citrate
buffer (pH 5.6) containing 0.3 mg of O-phenylenediamine/ml
and 0.03% H2O2. The reaction was stopped by
the addition of 4 N H2SO4. Optical density was
read at 490 nm. Controls were run in parallel on each plaque with a
series of dilutions from 1 to 3,000 pg/100 µl of IL-1 and IL-1ra.
quantification by RIA.
IL-1 concentrations were
determined by a radioimmunoassay (RIA) technique based on the
competition for a sheep antiserum against rabbit IL-1 between cold
IL-1 contained in the samples and a recombinant rabbit IL-1
labeled with 125I (the kit for RIA was from Endogen,
Cambridge, Mass.). A second antibody was used to immunoprecipitate the
first antibody binding to labeled or cold IL-1. Radioactivity was
read in a gamma counter (Beckman, Palo Alto, Calif.) and compared to a
standard curve.
, IL-1, and IL-1ra by RT-PCR in
tissues.
Total RNA was extracted from 100 mg of frozen Peyer's
patch tissue with guanidine thiocyanate (Bioprobe Systems, Montreuil sous Bois, France) as previously described (14) and
according to the manufacturer's instructions. Reverse transcription
(RT)-PCR was performed with specific primers which are detailed in
Table 1.
TABLE 1.
Primers used in RT-PCR
(21), primers were designed on two
different exons, based on sequence alignments between IL-1 and
IL-1 (GenBank accession no. M26295) obtained on Clustal V multiple sequence alignment (61) and with the PCRare program
(25). The cDNAs were synthesized after RQ1Dnase
treatment (2U; Promega France, Charbonnières, France) in a total
volume of 50 µl, using 10 µg of total RNAs and 0.5 µg of
oligo(dT)12-18 (Promega France) as a primer, 1.25 mM
deoxynucleoside triphosphate, 0.5 U of RNasin (Promega France), and 200 U of Moloney murine leukemia virus reverse transcriptase RNase-H minus
(Promega France) in the manufacturer's buffer for 1 h at 42°C.
PCRs were performed on a PHc-2 (Techne Inc., Rahway, N.J.). For a
100-µl reaction mixture, 5 µl of cDNA (serial dilutions), primers
(1 µM each), deoxynucleoside triphosphates (0.2 mM each),
MgCl2 (1.5 to 3 mM), and Eurobiotaq DNA polymerase (3.5 U;
Eurobio, Les Ulis, France) in the manufacturer's buffer were used. The
thermocycling protocol was as follows: 95°C for 3 min, then
x cycles (Table 1) of denaturation at 94°C for 45 s,
hybridization at annealing temperature (Table 1) for 1.5 min,
elongation at 72°C for 1.5 min, and a final incubation at 72°C for
7 min. The amplification products were resolved on a 1.5% agarose gel
containing 0.5 µg of ethidium bromide/ml and then transferred to a
nylon membrane (Hybond N+; Amersham, Courtaboeuf, France)
in 0.4 N NaOH and hybridized overnight at 60°C with the corresponding
oligonucleotide probes (Table 1) in Denhardt's reagent. The
oligonucleotides (5 pmol) were 5' end labeled with T4 polynucleotide
kinase (3 U; Pharmacia, Orsay, France) with 30 µCi of
[
-32P]ATP (5,000 Ci/mmol; Amersham) for 1 h at
37°C. Washing was performed two times for 30 min each time in 2×
saline sodium citrate-0.1% sodium dodecyl sulfate and two times for
30 min each time in 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate), and semiquantive analysis was achieved on a
PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.), using Image
Quant, comparing target molecule levels to -actin levels. We ensured
that measurements were performed in the exponential phase of the
reaction by varying cDNA dilutions and cycle number.
Statistical analysis.
We used the nonparametric Mann-Whitney
test (rank-sum test) for determination of the statistical significance
of differences between mean values. A probability of 
0.05 defined
this significance.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rabbits were laparotomized, and intestinal loops containing a Peyer's patch were ligated and injected either with the S. flexneri wild-type strain, M90T, or with the control strain, BS15, or with sterile saline. BS15 is a derivative of M90T in which the virulence plasmid pWR100 was cured and the strain was transfected with plasmid pRKB15 encoding AFR1, an adherence pilus of the rabbit-specific enteropathogenic E. coli strain RDEC-1 (13). AFR1 directs RDEC-1 primarily to M cells of the FAE and allows translocation of these bacteria to Peyer's patches (32). AFR1 allows translocation of BS15 through the FAE in a quantity similar to that of its invasive counterpart (56, 66). The tissue lesions observed in the present study were similar in their time of occurrence, quantity, and quality to those described in previous contributions, particularly one in which the M90T and BS15 strains were studied in parallel (56).
Infections with M90T and BS15 resulted in levels of bacterial invasion of Peyer's patches similar to those already shown (66), due to the AFR1 adhesin allowing a translocation efficiency through the FAE comparable to that caused by expression of the Shigella invasive phenotype. Two hours after infection, there were (2.1 ± 1.4) × 104 CFU/ml (mean ± standard deviation) in Peyer's patches infected with M90T and (2.8 ± 0.9) × 104 CFU/ml in infection with BS15. Four hours after infection, the numbers of CFU were (5.3 ± 1.9) × 104/ml for M90T and (6.2 ± 2.1) × 104/ml for BS15. Eight hours after infection, the numbers of CFU were (13.7 ± 5.8) × 105/ml for M90T and (15.6 ± 4.2) × 104 CFU/ml for BS15.
Blood from the mesenteric vein and Peyer's patch tissues was sampled
2, 4, and 8 h postinfection. IL-1, IL-1, and IL-1ra were
measured as described in Materials and Methods.
Concentration of IL-1 and IL-1 in plasma and Peyer's patch
tissue.
As shown in Fig. 1A, the
concentrations of IL-1 in plasma increased during the time course of
infection and were significantly higher in animals infected with M90T
at 2, 4, and 8 h after infection (1.08, 2.25 ± 0.45, and
2.65 ± 0.5 ng/ml, respectively) than in animals infected with
BS15 (0.04 ± 0.04, 0.13, and 0.92 ± 0.1 ng/ml,
respectively). As shown in Fig. 1B, consistent results were obtained
with tissue samples in which, at the three time periods, IL-1
concentrations were higher in animals infected with M90T (0.86 ± 0.1, 3.48 ± 0.7, and 3.63 ± 0.7 ng/ml, respectively) than
in animals infected with BS15 (0.04 ± 0.04, 1.53 ± 0.1, and 1.8 ± 0.18 ng/ml, respectively). In both plasma and tissue
samples, comparison of mean IL-1 concentrations at a given time
point in intestinal loops infected with M90T or BS15 showed a
P value of 0.05, thus confirming the statistical
significance. This series of results demonstrated that the invasive
strain M90T had the capacity, at each time point, to induce higher
IL-1 production than its noninvasive-adhesive counterpart.
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in plasma
samples were consistently higher in animals infected with M90T at 2, 4, and 8 h after infection (0.15 ± 0.15, 0.13 ± 0.01, and
0.23 ± 0.1 ng/ml, respectively) than in animals infected with BS15 (0.06 ± 0.01, 0.11, and 0.07 ± 0.01 ng/ml,
respectively). However, only the samples taken at 8 h showed a
statistically significant difference between M90T- and BS15-infected
loops, with a P value of <0.05. Consistent results, shown
in Fig. 1D, were obtained from tissue samples in which, at the three
time periods, IL-1 concentrations were higher in animals infected with M90T (1.65 ± 0.5, 4.18 ± 1.7, and 7.5 ± 1.7 ng/ml, respectively) than in animals infected with BS15 (0.62 ± 0.2, 2.04 ± 0.8, and 1.53 ± 0.4 ng/ml, respectively). At
the three time points, differences in mean IL-1 concentrations
between intestinal loops infected with M90T or BS15 appeared
statistically significant, with a P value of <0.05.
In the Peyer's patch tissue samples of control intestinal loops
infected with saline, levels of both IL-1 and IL-1 were stable
and very low at 2, 4, and 8 h compared to those corresponding to
infected tissues. After 8 h, the mean control values of tissue IL-1 and IL-1 were 0.58 ± 0.10 and 0.69 ± 0.22 ng/ml
of tissue suspension, respectively.
Taken together, these results demonstrate the rapid and specific
release of IL-1 and IL-1, first to lymphoid tissue and eventually
into circulation.
Concentration of IL-1ra in plasma and Peyer's patches. As shown in Fig. 1E, the concentration of IL-1ra in plasma samples was significantly lower after 2 and 4 h of infection in animals infected with M90T (1.26 ± 0.43 and 1.62 ± 0.57 ng/ml, respectively) than in animals infected by BS15 (4.97 ± 1.38 and 45.43 ± 12 ng/ml, respectively). At these two time points, differences in mean IL-1ra concentrations between loops infected with M90T or BS15 appeared statistically significant, with a P value of <0.05. After 8 h of infection, however, plasma and tissue concentrations of IL-1ra in M90T-infected loops had risen and even surpassed those measured in animals infected with BS15, in which IL-1ra concentrations had consistently decreased (13.68 ± 3.9 and 2.82 ng/ml, respectively). Similar trends were observed in tissue samples, as shown in Fig. 1F. After 2 and 4 h of infection, concentrations of IL-1ra were significantly lower in animals infected with M90T (82.5 ± 20 and 45.3 ± 11 ng/ml, respectively) than in animals infected with BS15 (179.8 ± 17.1 and 227.6 ± 64 ng/ml, respectively). At these two time points, differences in mean IL-1ra concentrations between loops infected with M90T or BS15 appeared statistically significant, with a P value of <0.05. Similarly, after 8 h of infection, IL-1ra concentrations had risen in M90T-infected samples and had reached concentrations even higher than those observed in BS15-infected tissues (229.22 ± 64 and 176.83 ± 30.7 ng/ml, respectively). This series of results shows a poor IL-1ra response in M90T-infected tissues compared to that in BS15-infected tissues during the first 4 h of infection. By 8 h, however, M90T-infected tissues had compensated for the differences and reached even higher concentrations of IL-1ra. In the Peyer's patch tissue samples from control intestinal loops infected with saline, levels of IL-1ra remained stable according to time. After 8 h, the mean value was 82.5 ± 18.8 ng/ml of tissue suspension.
Balance between IL-1ra and IL-1 in plasma and Peyer's patch
tissue.
Since IL-1 and IL-1 are proinflammatory and IL-1ra
is anti-inflammatory, we calculated the IL-1ra/IL-1 ratio for animals infected with M90T or BS15 at 2, 4, and 8 h postinfection in both plasma and tissue. Thus, high IL-1ra/IL-1 ratios would indicate inhibition of inflammation whereas low IL-1ra/IL-1 ratios are likely to
mean a strong inflammation. The results shown in Fig. 2 indicate a dramatic difference between
these ratios, depending on the infecting strain.
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ratio (Fig. 2B), in both plasma and tissues, is much
lower in animals infected with M90T after 2 and 4 h of infection
than in animals infected with BS15. The most striking difference was
observed after 4 h of infection, when the ratio was 34-fold lower
in plasma samples and 10-fold lower in tissue samples when M90T was the
infecting agent. After 8 h of infection, the ratios tended to equalize.
The difference between M90T and BS15 in the IL-1ra/IL-1 ratio was
even more striking (Fig. 2A). Two hours after infection, the ratios
were 12- and 47-fold lower in plasma and tissue samples, respectively,
when animals were infected with M90T. Four hours postinfection, the
ratios were 498- and 121-fold lower in plasma and tissue samples,
respectively. Here we also observed a trend toward equalization after
8 h of infection.
In the Peyer's patch tissue samples of control intestinal loops
infected with saline, the IL-1ra/IL-1 ratios remained stable. After
8 h, the IL-1ra/IL-1 ratio was 142 and the IL-1ra/IL-1 ratio
was 119.
These results indicate that at the early stage of Shigella
infection (i.e., after 2 and 4 h), M90T caused significantly
higher production of IL-1 and IL-1 than did BS15, as well as a
major imbalance in the IL-1ra/IL-1 ratio. Thus, the tissues are unable to increase the IL-1ra concentration to a level that would
counterbalance the increased IL-1.
Transcription of IL-1ra, IL-1, and IL-1 mRNAs.
We
analyzed the level of mRNA for IL-1ra, IL-1, and IL-1 to
determine whether there was transcriptional regulation during the
course of an infection by M90T or BS15. RT-PCR was used to analyze the
IL-1 and IL-1ra mRNAs. Figure 3 shows the
amount of mRNA for IL-1 (A), IL-1 (B), secreted IL-1ra (sIL-1ra)
(C), and intracellular IL-1ra (icIL-1ra) (D). The results are reported as the mean values of the numbers of pixels corresponding to
hybridization intensities as measured by the phosphorimager. The level
of mRNA was normalized to the signal given by -actin mRNA, which in
these experiments was standardized at a value of 500,000 pixels. In agreement with the concentrations of the translated products, Fig. 3A
and B show that the mRNA of IL-1 and IL-1 genes steadily increased with the duration of infection by M90T and BS15, although at
a significantly lower level at all time points in tissues infected by
BS15. Figure 3C and D show that transcriptional activities of the
sIL-1ra and icIL-1ra genes were consistently lower after 2 and 4 h
of infection in tissues infected by M90T than in those infected by
BS15.
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Immunostaining of Peyer's patch sections for LPS, macrophages,
IL-1, IL-1, and IL-1ra.
Figure
4 shows immunostainings performed on
serial sections of the same block taken from a Peyer's patch collected
after 4 h of infection, either with M90T (A to E) or with BS15 (F
to J). Immunostaining with an anti-LPS antibody (Fig. 4A) shows that M90T had already infected the FAE. Higher magnification (data not
shown) indicated that a large portion of the invading bacteria was
associated with M cells or M-cell pockets, as previously reported (56).
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-producing cells are present in both the FAE and
the dome of the Peyer's patch infected with M90T. Most of the positive cells correspond to cells expressing RAM11 in Fig. 4B. Conversely, there are fewer IL-1-producing cells in BS15 infection, where macrophages are recruited, than in M90T infection (Fig. 4H). A similar
observation can be made in Fig. 4D and I with cells producing IL-1,
which is massively expressed and probably released by the mononuclear
cells recruited in the course of M90T infection, whereas less of this
cytokine is expressed during BS15 infection. The pattern of IL-1
staining is less associated with individual cells than that of IL-1.
In agreement with the concentrations observed in tissues and draining
blood, weak labeling of IL-1ra is observed in both the FAE and the dome
of the Peyer's patch infected by M90T compared to that in BS15
infection. After 8 h of infection (data not shown), the numbers of
IL-1ra-producing cells were approximately equal in tissues infected
with M90T or BS15. In addition, epithelial cells, including M cells,
showed strong, homogeneous, and diffuse staining for IL-1 and
IL-1 (data not shown), indicating that epithelial cells had become
major players in the development of inflammation whereas at earlier
time points (i.e., 2 and 4 h) most IL-1 production took place in
macrophages and recruited monocytes.
Apoptosis in areas of infection and macrophage accumulation.
In order to analyze possible reasons for the low expression of IL-1ra
and massive release of IL-1 and IL-1 at 4 h after infection,
we looked for apoptosis in the areas of bacterial invasion and
macrophage accumulation in Peyer's patches infected either by M90T or
by BS15. As shown in Fig. 5A and C, the
dome area infected by invasive M90T was characterized by the presence
of a large number of apoptotic cells, the majority of which were likely
macrophages. On the other hand, as shown in Fig. 5B and D, very few
apoptotic cells were observed in areas of tissue infected by the
noninvasive strain BS15.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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IL-1 (IL-1 and IL-1) is a multipotent, primarily
inflammatory cytokine which affects almost any cell type and cooperates with other cytokines, chemokines, and a variety of other mediators. IL-1 is predominantly synthesized by activated macrophages but can also
be produced by members of the mononuclear phagocyte family, including
dendritic cells, and by other cell populations, such as B lymphocytes,
NK cells, endothelial cells, and even epithelial cells under particular
circumstances, such as bacterial invasion (17, 34).
Production of IL-1 is tightly regulated at the level of gene
expression, and its activity is controlled by a variety of molecules,
including surface receptors, such as IL-1RII, a nonagonist, decoy
receptor (57); soluble receptors, such as IL-1sRI and
IL-1sRII (6); and IL-1ra, a specific receptor antagonist (5, 7). This 23- to 25-kDa protein, which, like IL-1, is produced by monocytes/macrophages but also by PMN, fibroblasts, and
keratinocytes (4), binds to IL-1RI with affinity almost equal to that of IL-1 and IL-1 (19) but does not
transmit a signal (18). As a consequence, IL-1ra blocks IL-1
activity both in vitro and in vivo (4).
There are two major forms of the protein, sIL-1ra, which likely
corresponds to the above definition of a nonagonist competitor for
IL-1RI, particularly as an antagonist of the secreted form of IL-1,
and icIL-1ra, which shows an altered signal peptide. The physiological
function of icIL-1ra, however, is unclear (29).
In an increasing number of infectious and noninfectious clinical and
experimental situations, data indicate that IL-1ra may be essential for
the host defense against excessive, deleterious inflammation (20,
24, 26, 45, 50, 55). In IBD, and particularly in ulcerative
colitis, which shares several clinical and histopathological
characteristics with shigellosis, a significant imbalance of the
IL-1ra/IL-1 ratio, mostly caused by a decrease of IL-1ra concentrations
in intestinal tissues, was found at the acute phase of the disease
(11). The lowest IL-1ra/IL-1 ratios were observed in the
most severe cases. More recently, an increase in IL-1 and IL-1
was demonstrated in biopsy specimens from patients suffering acute
cases of Crohn's disease, ulcerative colitis, and other inflammatory
diseases of the colon. The IL-1ra/(IL-1 plus IL-1) ratio was
consistently decreased in these situations. In addition, a genetic
influence on the intensity of inflammation based on dysregulation of
the IL-1ra/IL-1 balance may account for an increase in disease severity
(3, 30, 52).
In patients at the acute and convalescent stages of S. flexneri and Shigella dysenteriae 1 infection,
immunohistochemistry of rectal samples shows a pattern of IL-1, IL-4,
IL-6, IL-8, tumor necrosis factor alpha, and gamma interferon
hyperproduction. Severe disease is associated with increased numbers of
IL-1-, IL-6-tumor necrosis factor alpha, and gamma
interferon-producing cells; IL-1 is essentially produced by
monocytes/macrophages, unlike IL-6 and IL-8, which are expressed by
epithelial cells (51). These data suggest that IL-1 is a key
factor in Shigella-induced intestinal inflammation. Our
previous observation that IL-1ra reduces intestinal inflammation in
experimental shigellosis in the rabbit ligated-loop model of
Shigella infection (55) represents the
experimental complement to these clinical observations and confirms the
key role of IL-1 in the initiation of inflammation. These data also suggest an imbalance in the IL-1ra/IL-1 ratio, as observed in IBD
(11). Moreover, in a model of rabbit immune complex colitis, IL-1 gene expression and synthesis occur early (i.e., at 4 h) in
the course of experimental disease and levels of IL-1 in tissues, which
are in the same order of magnitude as those observed here, correlate
with the degree of tissue inflammation, which is reduced by
administration of IL-1ra (15, 16).
The aim of this work was to follow the kinetics of IL-1, IL-1,
and IL-1ra in rabbit ligated loops infected by S. flexneri, both in infected tissues and in the mesenteric blood. We followed the
balance between these proinflammatory cytokines and their major
antagonist during the infection. Infections were carried out in loops
containing a Peyer's patch because the FAE that covers Peyer's
patches is the major site of Shigella invasion of intestinal tissues at early stages of incubation (i.e., at 2 to 4 h); thus, these areas are the major foci of the initiation of inflammation (47, 56, 59). In addition, rapid entry into these areas facilitates synchronization of the infectious process, whereas invasion
of villous areas of the intestinal epithelium is delayed and more
widespread in time (52a). In addition to invasive M90T, we
used a control mutant strain, BS15, a plasmidless, noninvasive mutant
of S. flexneri M90T which expresses the AFR1 rabbit-specific adherence pilus of the enteropathogenic E. coli RDEC-1
(13). This adhesin causes selective binding to the M-cell
surface, thus mediating the translocation of bacteria. In consequence,
M90T and BS15 reach similar numbers in infected Peyer's patches
(56, 66).
The results presented in this paper show an increase in production of
IL-1 during progression of the infection. At all time points, tissues
infected by the invasive strain M90T contained more IL-1 and IL-1
than those infected with the adhesive-noninvasive strain BS15. Tissue
concentrations of IL-1 in Peyer's patches infected by M90T were
roughly twofold greater than in those infected by BS15 at all time
points, and tissue concentrations of IL-1 were roughly two- to
fourfold greater, depending on the time point. This demonstrates that
invasive shigellae cause significantly greater release of IL-1 and
correlates with the observation that, in spite of its causing
significant influx of PMN in the domes of lymphoid follicles after
crossing the FAE, BS15 does not induce the extensive inflammatory
destruction observed with M90T (56). These results are also
in agreement with immunohistochemical observations of human rectal
samples (51). Higher production of IL-1 and IL-1 is
likely to reflect both higher expression of IL-1 in individual cells
and stronger recruitment of these producing cells to the infection
site. Accordingly, the number of transcripts for IL-1 and IL-1
followed a trend similar to that measured by the cytokines, as measured
by RT-PCR performed on tissue samples. Immunostaining experiments
carried out on infected Peyer's patch tissues clearly confirmed the
titration experiments; there were higher numbers of IL-1- and
IL-1-producing cells, as well as a greater amount of these cytokines
visible on the tissue sections of Peyer's patches infected with M90T
than on those infected with BS15.
Titrations carried out on the plasma obtained from the efferent
mesenteric blood samples taken from infected loops were in agreement
with these data, showing increasing concentrations of the two cytokines
during the infection. Interestingly, however, the ratio of plasma
IL-1 to tissue IL-1 was roughly 1 at all time points, whereas the
ratio of plasma IL-1 to tissue IL-1 varied between 0.1 and 0.2, thus appearing 10-fold lower. The latter observation was unexpected,
because in inflammatory situations, particularly in acute infections,
significant levels of circulating IL-1, but rarely of circulating
IL-1, are observed. This is essentially due to the property of
IL-1 of remaining primarily intracellular and being released mostly
in situations in which cytosolic leakage can occur, such as in the
event of cell lysis (58, 60). Conversely, mature IL-1 is
normally secreted by activated macrophages following cleavage of the
promolecule by caspase 1 (ICE) (9).
Macrophages from ICE knockout mice do not release mature IL-1 in
vitro (36, 38). The present observation is likely to reflect
a situation of massive killing of preactivated IL-1-producing cells in
areas where invading Shigella interacts with macrophages, particularly in the domes of follicular structures. This is precisely what was observed here with the early appearance (i.e., at 4 h) of
apoptotic cells, likely macrophages, in the apical domes of Peyer's
patches infected by M90T. Invasive Shigella causes
macrophage apoptosis both in vitro (67) and in vivo
(66). Apoptotic killing in vivo has been studied under
experimental conditions that were similar to those followed in the
course of this study. Under such conditions, after 8 h of
infection, the number of apoptotic cells is more than 50-fold greater
in follicles infected by M90T than in those infected by BS15
(66). IpaB is the major effector of apoptotic death by
activating ICE (12). This accounts for early release of
mature IL-1 by infected macrophages before they complete their cell
death program (31, 65), thereby initiating early inflammation in follicular zones (62, 63). The important
extracellular release of IL-1, which is reflected by its high
circulating titer in the mesenteric blood, may be explained by massive
killing of resident macrophages and recruited monocytes in the dome
area. This profound level of apoptosis overwhelms the phagocytic
clearance of apoptotic cells, thus allowing free extracellular IL-1
to be present in tissues and to subsequently pass into the circulation. Based on the above discussion, a model emerges in which not only the
difference in transcriptional and translational expression of IL-1
and IL-1 but also the complex intricacy of macrophage activation and
macrophage death caused by the invasive phenotype of M90T that makes
both forms of IL-1 more readily available to bind IL-1RI and activate
the large array of reactive cells present within the invaded zone can
account for the difference in induction capacity of tissue inflammation
and destruction observed between M90T and BS15.
Surprisingly, in addition to the higher expression of IL-1, M90T also
caused a decrease in the expression of IL-1ra in infected tissues at
4 h after infection in comparison with BS15. A twofold and a
fivefold decrease were observed at 2 and 4 h, respectively. At
8 h after infection, IL-1ra concentrations caught up, probably due
to massive recruitment of producing cells to infected zones. The plasma
concentrations reflected the tissue data, with a striking difference
observed at 4 h after infection; IL-1ra concentrations were
25-fold lower in mesenteric blood samples corresponding to Peyer's
patches infected with M90T than in those infected by BS15. These
differences were confirmed when the IL-1ra/IL-1 ratios were calculated.
In tissues infected by M90T, IL-1ra/IL-1 and IL-1ra/IL-1 ratios
were consistently less than 100, in contrast to tissues infected by
BS15, in which those ratios appeared constantly greater and, at 2 h, largely greater than 100.
The major differences appeared at the early 2- and 4-h time points of infection. Ratios in plasma samples dramatically accentuated these differences again at early stages of infection. The levels of mRNA clearly reflected the tissue levels of IL-1ra, with significantly lower activities after 2 and 4 h of infection in tissues infected by M90T than in those infected by BS15. This was observed both for sIL-1ra and icIL-1ra, thereby suggesting a common down-regulatory mechanism. It is possible that killing of macrophages at the early time points of infection accounts for this decrease.
It has previously been shown in human monocytes that LPS has the capacity to induce IL-1 and IL-1ra in the same cell (2). It is therefore possible that after releasing IL-1, the same macrophages and recruited monocytes are unable to compensate for their proinflammatory effect by producing enough IL-1ra because they are apoptotic. After 8 h of infection there is recruitment of PMN and circulating monocytes. Together, these cells control bacterial growth and increase the number of cells that produce IL-1ra, thereby restoring the IL-1ra-IL-1 balance.
It appears, therefore, that the early stage of Shigella infection is characterized by an imbalance between IL-1ra and IL-1 whose hypothetical mechanism is summarized in Fig. 6. In addition to the higher level of IL-1 production that characterizes the invasive phenotype, the lack of a proper balance between IL-1ra and IL-1 becomes another key feature of the development of severe inflammation. These studies certainly emphasize the need for experimental systems and analytical tools that would allow us to dissect the very early stages of infectious processes, particularly the early time points of the innate immune response, which are crucial for the development and subsequent healing of the disease lesions.
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ACKNOWLEDGMENTS |
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We thank Dana Philpott for her careful reading of the manuscript and Colette Jacquemin for editing. We also thank Armelle Phalipon and Juana Perdomo for their advice and constant interest in the work.
This work was supported in part by Biotec contract no. 97-C-0227 from the Ministère de l'Enseignement de la Recherche et de la Technologie and NIH grant AI42780.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité de Pathogénie Microbienne Moléculaire/Unité INSERM 389, Institut Pasteur, 25-28 Rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33) (0)1 45 68 83 42. Fax: (33) (0)1 45 68 89 53. E-mail: psanson{at}pasteur.fr.
Editor: J. R. McGhee
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, November 1999, p. 6056-6066, Vol. 67, No. 11
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