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Laboratoire de Microbiologie Génétique et Moléculaire Institut National de la Santé et de la Recherche Médicale U447,
Centre d’Immunologie et de Biologie Parasitaire, Institut National de la Santé et de la Recherche Médicale U167, and
Centre National de la Recherche Scientifique Unité de Recherche Associée 1854, Institut Pasteur de Lille, Lille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by T cells and NK cells. The
cDNA encoding IL-18 with its natural signal peptide was cloned under
control of the CMV promoter and injected into the skin of mice. A
single intradermal injection of this construction led to efficient in
vivo expression of IL-18 in cutaneous dermal cells and induced IFN-
mRNA production, indicating that it was produced in a biologically
active form. In addition, a massive cellular infiltrate was observed in
the skin 2 days after injection. When the mice were subsequently
infected with Mycobacterium bovis bacillus
Calmette-Guérin (BCG), they produced lower levels of anti-BCG
Abs than control animals. However, in contrast to their lowered humoral
immune response, the mice produced higher amounts of Ag-specific
IFN- after in vitro restimulation, as compared with the controls.
Therefore, injection of DNA encoding IL-18 into the skin modulates both
Ag-specific humoral and T cell responses upon mycobacterial infection.
It increases the Th1 type response, which may be particularly useful
for the development of new immunotherapeutic or immunoprotective
approaches against infections by intracellular parasites, such as
mycobacteria. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are associated with the generation of
cell-mediated immunity and resistance to intracellular parasites
(1), whereas Th2 cytokines favor the induction of humoral
immunity and resistance to extracellular parasites (2).
IL-18, first designated as IFN- inducing factor, is a newly
identified cytokine of the Th1 type (3), and the cDNAs
encoding murine and human IL-18 have recently been cloned (4, 5). Murine IL-18
(mIL-18)4 activates NK
cells, induces IFN- production by T cells stimulated with Con A,
anti-CD3 Abs, or IL-2, and promotes their proliferation (3, 4). IL-18 shares some biological activities with IL-12, although
both cytokines have different receptor-binding activities and signal
transduction pathways (6). It has been shown that IL-18,
but not IL-12, induces IL-2 production by Th1 clones stimulated with
immobilized anti-CD3 (6), and that IL-18-induced T
cell proliferation is IL-2-dependent (7). However, most
biological activities of IL-18 have been studied in vitro, and there is
only sparse information on the in vivo relevance of this cytokine in
immunological processes (8, 9). Therefore, we investigated
the immunomodulatory effects of IL-18 in the context of a mycobacterial
infection in vivo.
Protective immunity against mycobacterial infections is mediated by
interactions between specifically sensitized T cells and activated
macrophages (10). IFN- plays a crucial role in
antimycobacterial protection, as illustrated by the severe disseminated
form of tuberculosis in IFN- knockout mice (11, 12).
Infection with Mycobacterium tuberculosis also results in
secretion of IL-12, which is essential to generate protective immunity
through the induction of IFN- and the activation of Ag-specific
lymphocytes (13). Thus, cytokines prompting the immune
response to the development of the Th1 arm, able to induce
cell-mediated immunity, are of main interest for potential
immunotherapy against mycobacterial infections.
With the aim to study the function(s) and immunomodulatory effect(s) of
IL-18 in vivo, we used a DNA-based delivery approach. The mIL-18 cDNA
was cloned downstream of a CMV promoter and injected intradermally
(i.d.) into mice. The level of IFN- mRNA was assessed in the skin
following DNA administration, and histological studies revealed a
massive cellular infiltrate. Infection with Mycobacterium
bovis bacillus Calmette-Guérin (BCG) of mice injected with
the IL-18-encoding vector led to a reduction in the humoral immune
response against BCG, associated with a marked increase of Ag-specific
IFN- secretion. In addition to describing the immunological
properties of IL-18 in vivo, this study also demonstrates the utility
of this cytokine through DNA delivery, to tailor induction of specific
immune responses during infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cloning steps were performed in Escherichia coli XL1-Blue (Stratagene, La Jolla, CA). Expression of recombinant proteins was conducted in E. coli M15(pREP4) supplied by Qiagen (Courtaboeuf, France). BCG (vaccine strain 1173P2; World Health Organization, Stockholm, Sweden) was grown in Sauton medium (14) supplemented with 100 µg/ml ampicillin at 37°C using stationary tissue culture flasks. Vectors pQE-30 and pCR3.1-Uni were purchased from Qiagen and Invitrogen (San Diego, CA), respectively. Restriction enzymes, T4 DNA polymerase, and T4 DNA ligase were purchased from Boehringer Mannheim (Mannheim, Germany), and Vent DNA polymerase was purchased from New England Biolabs (Beverly, MA). All DNA manipulations were performed under standard conditions (15).
Construction of the IL-18 expression vectors
The cDNA encoding mIL-18 was obtained by RT-PCR on total RNA from J774 cells activated for 6 h with PMA (50 ng/ml), ionomycin (1 µg/ml), and LPS (1 µg/ml). Total RNA was extracted using RNAzol, according to the manufacturer’s recommendations (Bioprobe, Montreuil, France) and then subjected to RT-PCR using the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR kit (Perkin-Elmer, Roissy CDG, France) and the primers with the following sequences: 5'-CCAGATCTAACTTTGGCCGACTTCACTG-3' (P1) and 5'-AAGGTACCACCTAACTTTGATGTAAG-3' (P2), containing a BglII and an Asp718 sites (underlined). For expression in E. coli, the cDNA encoding the mature portion of mIL-18 was first amplified by PCR and inserted into pQE-30 to produce a mIL-18 fused at its N terminus to a histidine tag. The 491-bp PCR product was cut by BglII and Asp718 and cloned into BamHI/Asp718-restricted pQE-30, yielding pQE30::IL-18. For expression in eukaryotic cells, the complete IL-18-encoding gene was first amplified by RT-PCR using the primers P2 and P3 with the following sequence: 5'-CCCAGGAACCATGGCTGCCATGTC-3', containing a NcoI site (underlined). The resulting 598-bp fragment was inserted downstream of the CMV promoter into pCR3.1-Uni, using the Eukaryotic TA Cloning Kit-Unidirectional (Invitrogen) to generate pCR3.1::IL-18. pCR3.1 was generated by removing the 3'-protruding T nucleotides of pCR3.1-Uni with T4 DNA polymerase and religating the vector. For injection into mice, pCR3.1 and pCR3.1::IL-18 were prepared by alkaline lysis of the bacterial cultures, purified on CsCl gradient, and resuspended in apyrogenic injectable saline solution (Lavoisier, Paris, France).
Protein purification
Recombinant mIL-18 fused to the histidine tag (His6-IL-18) was produced in E. coli M15(pREP4) and purified from bacterial extracts, recovered 3 h after induction with 1 mM IPTG, using Co2+-based TALON Metal affinity chromatography under denaturing conditions (Clontech Laboratories, Palo Alto, CA). The eluted fractions were analyzed by SDS-PAGE (16) and Coomassie blue staining. Fractions containing His6-IL-18 were pooled and dialyzed against PBS and then subjected to preparative SDS-PAGE. His6-IL-18 was recovered from the gel by electroelution. The protein concentration was determined using the BCA Protein Assay Reagent kit (Pierce Europe, Breda, The Netherlands), according to the manufacturer’s instructions.
Preparation of anti-IL-18 antiserum
One volume of purified His6-IL-18 (350 µg) was combined with one volume of IFA (Sigma, St. Louis, MO) and injected i.d. into a male New Zealand white rabbit (Centre d’élevage et de Sélection J. Barroif, Tressin, France). The animal was boosted in the clavicula with 300 µg His6-IL-18 in IFA, 3 and 5 wk later. Serum was collected at various time points before and after the boost. Specificity of this antiserum was confirmed by immunoblotting against the purified protein.
Transient transfection in COS-1 cells
COS-1 cells were grown in DMEM (Life Technologies, Courbevoie, France) supplemented with 10% heat-inactivated FCS (Boehringer Mannheim), 2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). For transfection, cells were seeded in 6-well Falcon culture plates (Becton Dickinson, Meylan, France) at a concentration of 1–3 x 105 cells/well in 2 ml medium and grown at 37°C in a CO2 incubator until reaching 50–80% confluence. Medium was then removed and replaced by 1 ml of OPTI-MEM serum-free medium (Life Technologies). Cells were incubated for 6 h at 37°C in the presence of 1 µg of plasmid complexed to 3 µl of Exgen500 (Euromedex, Souffelweyersheim, France). The medium was then removed, and the transfected cells were incubated in complete medium containing FCS at 37°C for 48 h. The cells were then trypsinized and harvested by centrifugation at 1500 rpm for 10 min at room temperature. Lysates were obtained by resuspending the cell pellets in loading buffer (16). Expression of IL-18 was visualized by immunoblotting of culture lysates using the His6-IL-18 rabbit antiserum diluted 1/2000, and goat anti-rabbit alkaline phosphatase-conjugated Abs (Promega, Madison, WI) diluted 1/7000 in PBS containing Tween 20.
Plasmid administration and infection protocols
Six-week-old female BALB/c mice (Iffa Credo, l’Arbresle, France) were injected i.d. in the abdomen with 100 µl DNA solutions in a 1-ml syringe and a 27-gauge needle. For histochemical studies and RNA extractions, mice received a single injection of 50 µg of pCR3.1 or pCR3.1::IL-18. Mice were then sacrificed, and the skin from the injection site was carefully removed 1, 3, or 10 days after injection. For infection studies with BCG, groups of four to six mice were injected with three doses of 100 µg pCR3.1 or pCR3.1::IL-18 at days 1, 7, and 14. Mice were infected i.p. at day 3 with 5 x 105 or 5 x 106 viable BCG.
RNA extraction and RT-PCR analysis
The skin was cut into small pieces and immediately snap frozen.
Total RNA was extracted using RNAzol. Equal amounts of RNA (1 µg)
were reverse-transcribed with 200 U of Moloney murine leukemia virus
reverse transcriptase (Life Technologies), 4 U RNAsin (Promega), 50 ng
oligo(dT), 2 mM dNTP, 4 mM DTT in a final volume of 27 µl. The cDNAs
were then denatured by heating at 95°C before storage at -20°C.
PCR amplification was performed using primers for ß-actin
(5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTTTAGCACGCACTGTAATTCCTC-3'),
and IFN- (5'-GTCTGAAGTAACTATTTTAACAACTCAAG-3' and
5'-CGGTCAATGCTCCAGCTTGTTGGGTG-3'). The DNA was amplified by 35 cycles
for ß-actin, or 38 cycles for IFN- at an annealing temperature of
60°C, using a Perkin-Elmer DNA thermal cycler (Perkin-Elmer Cetus,
Saint-Quentin, France). In each case, a control synthesis reaction was
performed without RT to ensure that there was no contaminating DNA. The
PCR products were analyzed by agarose gel electrophoresis and ethidium
bromide staining.
Immunohistochemical analysis of the transfected skin
The skin samples were fixed in 4% paraformaldehyde before being dehydrated and embedded in paraffin. Tissue sections (4-µm thick) were cut, rehydrated and equilibrated with TBS (10 mM Tris-HCl (pH 7.4), 154 mM NaCl). For immunohistochemical staining, slides were incubated overnight at 4°C with rabbit anti-IL-18 serum or preimmune serum, diluted 1/1000 in TBS, washed extensively with TBS, and incubated for 2 h at room temperature with 1/200 goat alkaline phosphatase-conjugated anti-rabbit IgG (Sigma). After washing with TBS, Ab binding was visualized using the New Fuchsin Substrate System (Dako, Carpinteria, CA). The slides were counterstained with hematoxylin and examined by light microscopy.
Assay for IFN- production
Two weeks after the last DNA injection, inguinal, axillary, and
brachial lymph nodes (LN) were aseptically harvested. Cells were
isolated and resuspended in RPMI 1640 (Life Technologies) containing
10% heat inactivated FCS, 2 mM L-glutamine, 5 x
10-5 M 2-ME, and 1 mM sodium pyruvate. Cells
were cultured at a concentration of 5 x 105
cells/well in 96-well flat bottom culture plates in the absence or
presence of various concentrations of purified protein derivative (PPD;
kindly provided by Dr. K. Huygen, Institut Pasteur Bruxelles, Brussels,
Belgium). Culture supernatants were collected 4 days later, IFN-
production was measured by specific two-sites ELISA. The Ab pairs
(affinity-purified R5-6A2 mAb and biotinylated XMG1-2) were purchased
from PharMingen (San Diego, CA) and used as recommended by the
supplier. Optical densities at 492 nm were measured using a
multichannel spectrophotometer (Titertek multiskan MCC 1340). Results
are expressed as pg/ml of IFN-, calculated by reference to a
standard curve obtained with known amounts of recombinant purified
IFN- (PharMingen) diluted in culture medium.
ELISA
ELISA for the detection of anti-BCG Abs, as well as the preparation of BCG soluble Ags were conducted as described (17). Briefly, these Ags were prepared by sonication of BCG in PBS, followed by centrifugation at 10,000 x g for 30 min at 4°C, and then by centrifugation at 100,000 x g for 90 min at 4°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IL-18 expression plasmid, pCR3.1::IL-18, contains
the cDNA encoding full-length mIL-18 under the control of the CMV
promoter (Fig. 1
A). After
transient transfection of this plasmid into COS-1 cells, cytokine
production was detected using the anti-IL-18 rabbit antiserum. As
shown in Fig. 1B, an immunoreactive protein was present in
the lysate of cells transfected with pCR3.1::IL-18, but not
in that of cells transfected with pCR3.1, or of untransfected cells.
However, the immunoreactive protein migrated slower than the control
His6-IL-18 produced in E. coli.
Recently, it has been shown that the processing of IL-18 requires the
IL-1ß converting enzyme (ICE), and that IL-18 could only be
processed and activated in COS cells cotransfected with an
ICE-expressing vector (18, 19). Therefore, it is likely
that the slower electrophoretic mobility of IL-18 produced in COS-1
cells is due to a lack of signal peptide cleavage, rather than
posttranslational modification. Similar results were obtained when
Chinese hamster ovary and HeLa cells were transiently transfected with
pCR3.1::IL-18 (data not shown). Since processing of IL-18 by
ICE is necessary for its IFN--inducing activity (18),
it is likely that the cytokine produced in these transfected cells is
inactive.
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To investigate whether injection of pCR3.1::IL-18 can
result in in vivo synthesis of mIL-18, BALB/c mice were i.d. injected
with 50 µg of pCR3.1::IL-18 or of pCR3.1, and the skin was
removed 2 days later. Production of IL-18 was visualized by
immunohistochemistry using the anti-IL-18 rabbit antiserum. As
shown in Fig. 2A, IL-18 was
detected in dermal cells transfected with pCR3.1::IL-18. No
staining was observed with anti-IL-18 Abs on the skin of mice
injected with pCR3.1 (Fig. 2B), or with preimmune serum on
the pCR3.1::IL-18-transfected skin (Fig. 2C), or
on skin of mice injected with pCR3.1 (Fig. 2D). This
indicates that cutaneous transfection by pCR3.1::IL-18 led to
the production of IL-18 in the skin.
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Histological examination of the pCR3.1::IL-18-injected
skin revealed massive infiltration of cells in the dermis early after
DNA injection (Fig. 2
, A and C). This was not
seen in the dermis of mice injected with pCR3.1 (Fig. 2, B
and D). The cell infiltration was maximum 2 days
postinjection, disappearing almost completely after 10 days (data not
shown). This suggests that IL-18 was produced in its active form within
the skin, and therefore most likely processed by ICE in vivo.
Morphological analysis of infiltrating cells revealed that they mainly
consist of mononuclear cells, but we also detected granulocytes (data
not shown). The cellular infiltration was observed in three independent
experiments with three different DNA preparations.
IFN- response in the skin following injection of
pCR3.1::IL-18
To test whether injection of pCR3.1::IL-18 is able to
induce IFN- in vivo, RT-PCR was performed on RNA isolated from skin
of mice injected with pCR3.1::IL-18 or pCR3.1 1, 3, and 10
days after DNA inoculation. As shown in Fig. 3, high levels of IFN--specific mRNA
were present in the skin 1, 3, and 10 days after injection with
pCR3.1::IL-18. In contrast, in skin injected with pCR3.1,
IFN--specific mRNA were detectable only 1 day after DNA inoculation
and at lower amounts. These results confirm that the IL-18 produced by
cutaneous-transfected cells is biologically functional and able to
activate cells for the expression of cytokines. The low nonspecific,
transient IFN- production in the skin of mice injected with pCR3.1
is probably due to DNA immunostimulatory motifs present in the plasmid
backbone (20, 21, 22).
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A Th1 response is known to be pivotal for controlling
mycobacterial infections (11, 12, 13). IL-18 has been shown to
synergize with IL-12 for the induction of IFN- in mice
(23), which may modulate both the humoral and cellular
immune responses to infections or administered Ags. To examine the
effect of IL-18 on the cellular immune response against BCG, mice were
infected i. p. with two doses of live BCG (5 x
105 or 5 x 106) after
injection of pCR3.1::IL-18 or pCR3.1. Skin-draining LN cells
were then removed and incubated with various concentrations of PPD. As
shown in Fig. 4, an important
dose-dependent IFN- production was detected in the cells of mice
having received BCG together with pCR3.1::IL-18, compared
with cells of mice that had received BCG together with the control
plasmid. Consistent with previous observations that lower doses of BCG
often induce higher levels of IFN- than higher doses, the cells of
mice infected with 5 x 105 BCG produced
somewhat more IFN- than those of mice infected with 5 x
106 BCG. No IFN- was detected in mice injected
with either plasmid alone, indicating that the production of IFN- in
mice infected with BCG was specifically induced after restimulation
with PPD. No IL-4 production could be detected in the supernatants of
these cell cultures (data not shown).
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), indicating that IL-18 negatively
affected Ag-specific Ab production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or IL-12, or Tcell costimulatory
molecules has been demonstrated to result in marked enhancement of both
specific T helper and cytotoxic responses (24, 25, 26, 27). It is
likely that triggering the cytokine cascade through one of its earliest
steps may have the most pronounced and long-lasting effects on the T
cell responses. IL-18 is a recently identified cytokine that appears to
act at a very early step in the T cell activation cascade. It induces
IFN-, especially in synergy with IL-12. The production of IFN-
and the subsequent effect on T cell activation is particularly
important for the protection against intracellular parasites, such as
mycobacteria. In most cases, M. tuberculosis infections are
well controlled by the cell-mediated host immune system. However, in
10% of the cases, disease develops, probably reflecting an
insufficient T cell response. Tuberculosis often occurs after
reactivation of dormant bacilli, suggesting that immunomodulation of
infected subjects may help preventing reactivation disease. Infection
with mycobacteria usually results in the secretion of large amounts of
IL-12 (28), leading to IFN- production and T cell
activation. Therefore, we investigated whether the injection of an
IL-18-encoding plasmid could enhance the T cell response to
mycobacterial Ags in mice infected with BCG, used as a model
system. In this study, we show that the injection of an IL-18-encoding plasmid into the skin of mice results in local IL-18 production and induces a prominent and disseminated cell infiltrate at the injection site. The induction of massive infiltration of inflammatory cells in mice injected with purified recombinant IL-18 has been described as one of the hallmarks of IL-18 activities (8), confirming its role in inflammatory processes (3, 4, 29). These observations therefore indicate that the injection of the IL-18-encoding plasmid results in the production of biologically active IL-18. Biological activity of this cytokine is only expressed when the preprotein is processed into the mature form. This maturation requires ICE (18, 19). Processing did not occur in transiently transfected COS, Chinese hamster ovary, or HeLa cells, most likely because ICE is not coproduced in these cells. Since IL-18 appeared to be biologically active in the skin injected with the IL-18-encoding plasmid, it was most likely produced in cells that also produce ICE. We have indeed found ICE mRNA present in the skin of injected animals (data not shown). Ariizumi et al. (30) have shown that murine epidermal-derived dendritic cell lines express ICE in a biologically active form. Furthermore, dendritic cells, as well as keratinocytes and fibroblasts, are readily transfected upon i.d. injection of plasmid DNA (31). We can therefore hypothesize that dendritic cells may be at least one of the cell types that expresses the IL-18 cDNA upon injection of the IL-18-encoding plasmid into the skin. The coexpression of biologically active ICE within these cells may then result in the production of IL-18 in its mature form.
The biological activity of IL-18 in the plasmid-injected skin was
confirmed by the induction of IFN- mRNA within the skin. Substantial
amounts of IFN- mRNA were produced in the skin of mice injected with
the IL-18-encoding plasmid. Interestingly, small, transient amounts of
IFN- mRNA were also produced in the skin of mice injected with the
control plasmid, suggesting that the plasmid itself may have some
IFN--inducing activity. Recent studies have shown that plasmid DNA
from bacterial origin may contain immunostimulatory sequences (ISS),
characterized by a CpG motif, that exert immunomodulatory effects on B
cells, T cells, or macrophages (21, 22). These effects are
likely to be mediated by the production of cytokines, including
IFN-, IL-12, and TNF-
(20). Inspection of the
sequences of the IL-18-encoding plasmid and the control plasmid
indicated that both contain six ISS (data not shown). Among these six
CpG-containing hexamers, four have the sequence 5'-GACGTC-3' and two
the sequence 5'-AACGTT-3'. Both are sequences known to induce IFN-
secretion (20). These observations suggest that ISS may
contribute to the IFN- production in the skin of the injected mice.
However, the IFN- production in the skin of mice injected with the
control plasmid was only weak and transient, whereas IFN- production
in the skin of the mice injected with the IL-18-encoding plasmid was
much stronger and lasted for at least 10 days, indicating that it was
induced by active IL-18 expressed by transfected cutaneous cells.
Most importantly, the injection of the IL-18-encoding plasmid affected
Ab and cellular responses to mycobacterial Ags after infection with
BCG. Administration of the IL-18-encoding plasmid led to increased in
vitro PPD-dependent IFN- production, whereas no IL-4 production
could be detected. On the other hand, the level of total soluble
antimycobacterial IgG was decreased in the presence of the IL-18
plasmid. Thus, IFN- produced by IL-18-activated Th1 cells may have
selectively inhibited Th2 cells, thereby contributing to the absence of
IL-4 production and decreasing the Ab response against BCG. This latter
observation is in line with previous studies showing that injection of
Th1 cytokine-expressing plasmids may result in the reduction of a
specific Ab response (24, 27). Flow cytometry analysis
using Abs specific for B220+,
CD4+, and CD8+ cells
indicated that the proportion of these different cell types were
similar, regardless whether the mice were injected with the
IL-18-encoding plasmid or with the control plasmid (data not shown).
This suggests that the impaired humoral and the enhanced T cell
responses against mycobacteria were not caused by a dysregulation of
the B cell/T cell balance or of the
CD4+/CD8+ ratio, but rather
by the capacity of IL-18 to induce T cell activation, especially of Th1
cells.
It remains to be investigated, whether IL-18 exerts its inflammatory
effect directly or via the induction of proinflammatory cytokines, such
as TNF-, IL-1ß, or IL-8. Human IL-18 has been found to enhance
GM-CSF production in Con A-stimulated PBMC and to inhibit the release
of IL-10 (5). It might thereby enhance the accessory
functions of dendritic cells, such as Langerhans cells, since GM-CSF is
known to be involved in the growth and differentiation of Langerhans
cells (32, 33), whereas IL-10 inhibits the maturation of
these cells (34). Thus, IL-18 might indirectly enhance the
maturation and activation of Langerhans cells, which, in turn, may
contribute to the inflammatory reactions observed.
Regardless the mechanism, the results reported in this study indicate that, delivery of IL-18 through DNA administration can modulate a specific immune response toward a Th1 response in the course of a bacterial infection. This may be particularly useful for the control or prevention of infection by intracellular parasites, such as mycobacteria, or perhaps HIV. With the emergence of tuberculosis in HIV-infected individuals and of multidrug-resistant strains of M. tuberculosis, immunotherapy with recombinant cytokines in adjunct to chemotherapy is currently being considered for treatment of certain cases of tuberculosis (35, 36). In vivo expression of cytokines using DNA delivery systems may help to define the cytokines that are most useful for immunotherapy or to induce protective immunity against tuberculosis. Of course, before IL-18-encoding plasmid DNA can be used in clinical settings, a number of points need to be considered, especially with respect to the safety of this approach. In addition to the concerns regarding DNA vaccination in general (reviewed in Ref. 37), the effects of the important cellular infiltrates after IL-18-DNA injection will have to be addressed. However, we observed that these infiltrates were localized to the site of injection and transient, with a maximum 2 days after injection, and a complete disappearance 10 days after injection. In addition, by using different DNA delivery methods and by using different doses, we hope that optimized conditions can be found to induce maximal immunomodulatory effects with minimal inflammatory effects. If safety issues can be satisfactorily resolved, the use of DNA encoding cytokines, such as IL-18, has the advantage of a more sustained, albeit transient, presence of the cytokine at the site of injection, as compared with the injection of the purified protein. This would circumvent the short half-life of recombinant IL-18 and the side effects due to the administration of repetitive, high doses.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology, School of Microbiological, Immunological and Virological Sciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K.
3 Address correspondence to Dr. Camille Locht, Laboratoire de Microbiologie Génétique et Moléculaire Institut National de la Santé et de la Recherche Médicale U447, Institut Pasteur de Lille, 1 rue du Prof. Calmette, F-59019 Lille Cedex, France. E-mail address:
4 Abbreviations used in this paper: mIL-18, murine IL-18; BCG, bacillus Calmette-Guérin; ICE, IL-1ß converting enzyme; i.d., intradermal; LN, lymph nodes; PPD, purified protein derivative.
Received for publication March 15, 1999. Accepted for publication June 30, 1999.
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 M. Umemura, H. Nishimura, K. Hirose, T. Matsuguchi, and Y. Yoshikai Overexpression of IL-15 In Vivo Enhances Protection Against Mycobacterium bovis Bacillus Calmette-Guerin Infection Via Augmentation of NK and T Cytotoxic 1 Responses J. Immunol., July 15, 2001; 167(2): 946 - 956. [Abstract] [Full Text] [PDF]
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 H.-R. Jiang, X.-q. Wei, W. Niedbala, L. Lumsden, F. Y. Liew, and J. V. Forrester IL-18 Not Required for IRBP Peptide-Induced EAU: Studies in Gene-Deficient Mice Invest. Ophthalmol. Vis. Sci., January 1, 2001; 42(1): 177 - 182. [Abstract] [Full Text]
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A. Mencacci, A. Bacci, E. Cenci, C. Montagnoli, S. Fiorucci, A. Casagrande, R. A. Flavell, F. Bistoni, and L. Romani Interleukin 18 Restores Defective Th1 Immunity to Candida albicans in Caspase 1-Deficient Mice Infect. Immun., September 1, 2000; 68(9): 5126 - 5131. [Abstract] [Full Text] [PDF]
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 M. M. Gherardi, J. C. Ramírez, and M. Esteban Interleukin-12 (IL-12) Enhancement of the Cellular Immune Response against Human Immunodeficiency Virus Type 1 Env Antigen in a DNA Prime/Vaccinia Virus Boost Vaccine Regimen Is Time and Dose Dependent: Suppressive Effects of IL-12 Boost Are Mediated by Nitric Oxide J. Virol., July 15, 2000; 74(14): 6278 - 6286. [Abstract] [Full Text]
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 G. YAMADA, N. SHIJUBO, K. SHIGEHARA, H. OKAMURA, M. KURIMOTO, and S. ABE Increased Levels of Circulating Interleukin-18 in Patients with Advanced Tuberculosis Am. J. Respir. Crit. Care Med., June 1, 2000; 161(6): 1786 - 1789. [Abstract] [Full Text]
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