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*
Division of Dermatology and
Department of Microbiology and Immunology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095;
Division of Rheumatology and Immunology, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115;
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Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Japan;
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Fujisaki Institute, Hayashibara Biochemical Labs, Fujisaki, Okayama, Japan; and
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Section of Dermatology, University of Southern California School of Medicine, Los Angeles, CA 90033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in tuberculoid patients, but not
lepromatous patients, while IL-4 production was not induced by IL-18.
Anti-IL-12 partially inhibited M. leprae-induced release
of IFN- in the presence of IL-18, suggesting a combined effect of
IL-12 and IL-18 in promoting M. leprae-specific type 1
responses. IL-18 enhanced M. leprae-induced IFN-
production rapidly (24 h) by NK cells and in a more sustained manner (5
days) by T cells. Finally, IL-18 directly induced IFN- production
from mycobacteria-reactive T cell clones. These results suggest that
IL-18 induces type 1 cytokine responses in the host defense against
intracellular infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and are generally associated with resistance to intracellular
infection, whereas type 2 T cells release IL-4, which is associated
with progressive disease (1, 2, 3). In this context, cells from the innate
immune response, dendritic cells, monocytes, and NK cells, shape the
nature of the subsequent adaptive T cell response by influencing the
cytokine pattern (4). In particular, IL-12, a cytokine produced by
dendritic cells and monocytes, plays an obligatory role in the
generation of type 1 cells (5, 6), promotes NK cell stimulation (7),
and induces IFN- production from NK and T cells (8, 9).
Recently, IL-18 has been shown to influence both the innate and
adaptive immune response by enhancing type 1 cytokine responses of NK
and T cells, respectively (10, 11). Macrophages have been shown to
produce IL-18 as well as IL-12 (12). IL-18 has been found to have a
variety of biological actions, including stimulating proliferation of
activated T cells, enhancing NK cell activity, and inducing type 1
cytokine responses (11, 12, 13, 14). Several investigators have demonstrated
that IL-12 has a synergistic effect with IL-18 on the production of
IFN- by anti-CD3-activated T cells (11, 14) and
anti-CD40-activated B cells (15). Moreover, a recent study has
shown that exogenously administrated IL-12 and IL-18 acted
synergistically to protect mice against cryptococcal infection (16).
Additional studies have indicated a role for IL-18 in immune responses
to infection, both in mouse models and human disease (17, 18, 19, 20, 21).
Leprosy provides an ideal model to investigate the regulation and role of IL-18 in human infectious disease, because the various clinical manifestations of leprosy have been shown to lie on an immunological spectrum, according to the level of cell-mediated immunity (CMI)3 to the pathogen, Mycobacterium leprae (22). At one extreme, tuberculoid patients are able to restrict the growth of the pathogen, mount strong T cell responses to M. leprae, and locally produce the type 1 cytokine pattern (2, 3). In contrast, lepromatous patients manifest disseminated infection, their T cells weakly respond to M. leprae, and their lesions express the type 2 cytokines, typical of humoral responses and suppression of CMI (3, 23). In this study, we provide evidence that IL-18 contributes to CMI in human infectious disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients with leprosy were evaluated at the Los Angeles County Hansen’s Disease Clinic and classified according to the criteria of Ridley and Jopling (22). Peripheral blood was collected in heparinized tubes from patients with tuberculoid (T-Lep) and lepromatous (L-Lep) leprosy. After receiving informed consent, skin biopsies from leprosy patients were obtained, embedded in OCT medium (Ames, Elkhart, IN), snap frozen in liquid nitrogen, and stored at -70°C.
M. leprae
M. leprae was provided by Dr. Patrick Brennan (Colorado State University, Ft. Collins, CO) and prepared by probe sonication (24). The level of LPS in the M. leprae sonicate was measured quantitatively with the Limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD) and found to be <1.0 endotoxin U/mg M. leprae sonicate. A similar extract was prepared from the virulent Mycobacterium tuberculosis strain H37Rv. Stimulation of cells throughout the present study was performed with these sonicated preparations of mycobacteria.
Cell preparations
PBMCs were isolated from heparinized blood by density gradient centrifugation on Ficoll-Paque (Pharmacia, Piscataway, NJ) and cultured in 24-well plates at 106 cell/ml with RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 2 mM glutamine (Life Technologies), 0.1 mM sodium pyruvate (Life Technologies), and 10% of heat-inactivated FBS (HyClone Laboratories, Logan, UT) for 24 or 48 h according to the experiment. Human rIL-18 (Hayashibara Biochemical Laboratories, Okayama, Japan) was obtained from Escherichia coli and purified by chromatography as described (12). Human rIL-12 (a generous gift from Hoffman-La Roche, Nutley, NJ) was added to some cultures at final concentration of 1 nM.
To determine which cell population(s) respond to IL-18,
CD3+ or CD56+ cells were depleted from PBMCs by
two rounds of negative selection using magnetic beads (Dynal, Oslo,
Norway). The negative cells were washed, counted, and depletion was
evaluated by flow cytometry. Cells were then cultured in 96-well plates
at 106 cell/ml in RPMI 1640 supplemented as described
above, in the presence of M. leprae with or without
increasing concentrations of IL-18 as shown in the figures. After
24 h, cell-free supernatants were collected, and IFN-
concentration was determined by ELISA.
Purified monocytes were obtained as described previously (25). Briefly, blood was collected into heparinized syringes containing Plasmagel (Laboratoire Roger Bellon, Neuille, France), and red cells were permitted to sediment to the bottom of the syringe. The cellular supernatant was collected, and the leukocytes were incubated in hypertonic (1.54 M NaCl) PBS. The monocytes resist a change in density in the presence of high salt concentrations as compared with other leukocytes that rapidly become denser under such conditions. Monocytes were harvested by density centrifugation in hypertonic Ficoll-Hypaque (Pharmacia). This protocol routinely gives a monocyte purity of 73 ± 8% monocytes and a yield of 3.4 x 105 monocytes/ml blood. Purified monocytes (1 x 106 cell/ml; 79.6% monocytes) were stimulated in the presence or absence of M. leprae for 16 h before RNA isolation.
PBMCs from T-Lep and L-Lep patients were isolated, and adherent cells were prepared as previously described (26). Adherent cells were plated (1 x 106/ml) with M. leprae (5 µg/ml) or without Ag at 37°C in a CO2 incubator with RPMI 1640 and 10% FCS for 3, 16, and 36 h, and total RNA was isolated.
The human myelomonocytic leukemia cell line U-937 was maintained in culture in RPMI 1640 supplemented with 10% FCS. Cells were cultured in 24-well plated at 106 cell/ml with M. leprae, M. tuberculosis, or LPS.
Detection of IL-18 mRNA
PCR for IL-18 mRNA in leprosy lesions and purified monocytes was performed as described (3, 26, 27). RNA was isolated by guanidinium isothiocyanate lysis, phenol-chloroform extraction, and ethanol precipitation. DNA was removed using RNase-free DNase (10 U; Promega, Madison, WI). First strand cDNA was synthesized using Superscript reverse transcriptase (Life Technologies Life Science, Bethesda, MD) and oligo dT priming (Pharmacia). cDNA samples were amplified with IL-18-specific PCR primers and Taq polymerase (Perkin-Elmer, Norwalk, CT) in a DNA thermocycler (Perkin-Elmer). The PCR conditions consisted of 40 cycles of denaturation (94°C for 30 s) and annealing-extension (65°C for 45 s). For comparison of IL-18 mRNA levels among different samples, cDNA concentrations were normalized to yield equivalent ß-actin PCR products. PCR products were electrophoresed on 1.5% agarose gels, transferred to nylon membranes (Amersham), probed with a 32P-labeled IL-18 oligonucleotide internal to the PCR primers, and visualized by autoradiography. The sequences of primer pairs, 5' and 3', were as follows: 5'-GCTTGAATCTAAATTATCAAGTC, 3'-GAAGATTCAAATTGCATCTTAT. The IL-18 probe sequence was as follows (5' to 3'): TGACTGTAGAGATAATGCACCCCGGACC.
Blocking experiments
PBMCs from patients were cultured with M. leprae
alone or in the presence of neutralizing anti-IL-18 mAb (10
µg/ml), anti-IL-12 mAb (10 µg/ml;Endogen, Cambridge, MA), or
isotype control mouse IgG (Becton Dickinson, San Jose, CA) for 48
h and then assayed for IFN- production by ELISA. In these series of
experiments, cells were incubated with the neutralizing mAb for 2
h before the addition of the Ag. Anti-IL-18 mAb (BALB/c mouse IgG1
,
purity 90%) was obtained from Hayashibara Biochemical Laboratories
(mAb human IFN--inducing factor (HuIGIF) 125-2H) and used at 10
µg/ml according to the reported activity (1 µg/ml mAb-HuIGIF 125-2H
neutralizes 5ng/ml rHuIGIF).
T cell clones and T cell lines
The T cell clones and cell lines used in this study have been
described (28, 29). 
T cells (HF.2, 1 x 105)
were plated in microtiter plates and stimulated with isopentenyl
pyrophosphate (IPP; 3 µM; Sigma, St. Louis, MO) in the absence of
APC. We used the HF.2 T cell clone that specifically recognizes
IPP, a metabolite found in prokaryotic and eukaryotic cells (30). The
CD1b-restricted T cell line LDN4 was cultured with Ag lipoarabinomannan
in the presence of CD1+ APC as described (29). rIL-18 was
added to the T cells or to the double-negative T cells at
different concentrations as indicated in the legend to Fig. 9
. After
overnight incubation, supernatants were collected, and IFN- levels
were measured by ELISA.
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The concentration of IL-18 in the culture supernatants was measured by ELISA as described (31). In brief, 96-well ELISA plates (Corning Glass Works, Corning, NY) were coated with mAb 125-2H (20 µg/ml in PBS) at room temperature (RT) for 3 h and blocked with PBS containing 1% BSA at 4°C overnight. Standard human IL-18 (Hayashibara Biochemical Laboratories) (12) and samples were added and incubated at RT for 2 h. Peroxidase-conjugated anti-IL-18 (159-12B, 0.5 µg/ml) was added to each well, and the plate was incubated at RT for 2 h. Peroxidase substrate solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and the plates were read in an ELISA reader (Cambridge Technology, Watertown, MA) at a wavelength of 405 nm. Washing steps (PBS containing 0.1% BSA and 0.05% Tween 20) were included between each step of the ELISA. A standard curve was made by plotting the duplicate readings for the standards vs the concentration of the standards, and regression analysis was applied. The IL-18 concentration of each sample was calculated by regression analysis using the mean absorbance (average of triplicate readings) of the sample.
IFN- (Endogen) ELISA was performed according to the manufacturer’s
instructions. Briefly, flat-bottom 96-well microtiter plates were
coated with mouse anti-human IFN- mAb (M-700A; Endogen) at 5 µg/ml
in sodium carbonate buffer, pH 9.6, and incubated overnight at 4°C,
followed by blocking with PBS containing 1% BSA for 1 h at RT.
Samples and IFN- standard (human rIFN-, R-IFN--50; Endogen)
were serially diluted and incubated at RT for 3 h. Subsequently,
biotinylated anti-IFN- mAb (M-701-B; Endogen) was added at 2
µg/ml for 1 h at RT. Avidin-peroxidase conjugate (A-3151; Sigma)
was then added at 2 µg/ml for 30 min at RT. Peroxidase substrate
solution (Kirkegaard & Perry Laboratories) was added, and the plates
were read in an ELISA reader (Cambridge Technology) at a wavelength of
405 nm. Washing steps (PBS containing 0.1% BSA and 0.05% Tween 20)
were included between each step of the ELISA. A standard curve was made
and regression analysis was applied. The IFN- concentration of each
sample was calculated by regression analysis using the mean absorbance
(average of triplicate readings) of the sample. The sensitivity of this
assay was 10 pg/ml.
Intracellular analysis of cytokine production
Intracellular cytokine staining was used to determine the
IFN- production at the single cell level as previously described
(32). Briefly, to induce the intracellular accumulation of newly
synthesized proteins, monensin (2 µM; Calbiochem, La Jolla, CA) was
added for 2 h to the cells in culture. Cells were then harvested
and stained for surface expression with mAbs specific for CD56 (Becton
Dickinson) and CD3 (Becton Dickinson). After washing with PBS-2% FCS,
the cells were fixed with 4% paraformaldehyde in PBS for 30 min at RT.
Cells were washed with PBS-2% FCS and permeabilized with 0.5% (w/v)
saponin (Sigma) in PBS during 30 min at RT. PE-conjugated
anti-IFN- (used at the manufacturer’s recommended
concentrations; PharMingen, San Diego, CA) was added to the
permeabilized cells and allowed to bind for 30 min. Cells were then
washed with PBS-0.5% saponin and finally with PBS/2% FCS to allow
membrane closure. Samples were analyzed on a FACScan flow cytometer,
and data were analyzed using LYSIS software (Becton Dickinson).
Negative control samples were incubated with irrelevant isotype-matched
Abs in parallel with all experimental samples.
Statistical analysis
Nonparametric statistical analysis by the Signed rank test for paired samples was used for comparison of cells after culture. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the relative distribution of IL-18 in leprosy
patients, we performed PCR on RNA derived from leprosy lesions. cDNAs
were synthesized and were normalized to amounts of ß-actin PCR
products as a measure of total cellular RNA. Our results showed that
IL-18 mRNA could be detected in the majority of the T-Lep patients (7
of 10), whereas it was undetectable in the L-Lep patients (Fig. 1A). Therefore, the
distribution of IL-18 in leprosy, parallels the level of CMI to
M. leprae.
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B).
It has been shown that IL-18 mRNA could be detected in the total RNA of
human unstimulated PBMCs, with no significant changes of IL-18 mRNA
expression after mitogen stimulation (12). Because human IL-18 is known
to be produced by cells of the monocyte lineage (12), we investigated
whether M. leprae could stimulate detectable levels of IL-18
mRNA in purified human monocytes compared with nonstimulated cells.
RT-PCR analysis demonstrated a significant induction of IL-18 mRNA
expression in the presence of M. leprae (Fig. 2A).
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B). These results correlate with the
expression of IL-18 mRNA in lesions. M. leprae-stimulated IL-18 release
We next investigated whether IL-18 protein was released upon
stimulation with M. leprae. To investigate this possibility,
we stimulated human monocytic cell lines, monocytes, and PBMCs with LPS
or with mycobacteria. Stimulation of the monocytic cell line U937 with
mycobacteria showed that IL-18 was barely detectable (limit of
detection = 10 pg/ml) in cultures grown in the absence of
mycobacteria. In contrast, M. leprae and M.
tuberculosis stimulated detectable IL-18 protein, while
stimulation with LPS also induced IL-18 (Fig. 2C). M.
leprae also induced considerable levels of IL-18 after stimulation
of THP-1 cells (THP-1 mycobacterial stimulation, 450 pg/ml). While
specific Ag stimulation of human cell lines induced considerable levels
of IL-18 protein, low levels of IL-18 were detected after stimulation
of either monocytes (LPS stimulation, 90 pg/ml; mycobacterial
stimulation, 30 pg/ml) or PBMCs (LPS stimulation, 25 pg/ml;
mycobacterial stimulation, 35 pg/ml). Other investigators have studied
the production of IL-18 after microbial infection (21, 33) with
variable results; the low level of IL-18 protein may be explained by
absorption by T and B cells in the culture (20).
In a separate set of experiments, neutralizing anti-IL-18 Abs were
used to determine whether M. leprae-induced IFN-
production was dependent on endogenous IL-18 production. PBMCs from
T-Lep patients were stimulated with M. leprae in the
presence or absence of neutralizing anti-IL-18 Ab or an isotype
control Ab. As shown in Fig. 3A, the IFN- levels induced
in response to Ag were inhibited by 80–99% in T-Lep patients,
demonstrating that endogenous IL-18 production contributes to M.
leprae-induced IFN- production.
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It has been reported that human IL-18 showed significant
IFN--inducing activity when human PBMCs were stimulated with
anti-CD3, ConA, or IL-2, but not when unstimulated PBMCs were used
(12). We next examined whether IL-18 could modify the response of human
PBMCs to a specific Ag. To address this question, we stimulated PBMCs
from a large group of leprosy patients in the presence or absence of
rIL-18. Fig. 3B illustrates that IL-18 significantly
increased M. leprae-induced IFN- production from T-Lep
patients (p < 0.001). No effect of IL-18 on
IFN- production was observed in the absence of Ag (data not shown).
Given the dependence of M. leprae-induced T cell
proliferation on IL-12, we investigated whether IL-18 could enhance
Ag-induced T cell responses in L-Lep patients. Our results showed that,
in marked contrast with the effect of IL-12 on M.
leprae-specific T cell responses, IL-18 did not augment Ag-induced
IFN- production from L-Lep patients (Fig. 3B). The
addition of rIL-18 had no effect on IL-4 production, either from T-Lep
or from L-Lep patients (data not shown).
In an attempt to investigate the interaction of IL-18 and IL-12 in the
immune response to M. leprae, we first studied the effect of
neutralizing anti-IL-12 Ab on the M. leprae-induced
IFN- production in the presence of IL-18. PBMCs from T-Lep patients
were stimulated with M. leprae and IL-18 in the presence or
absence of anti-IL-12 Ab or an isotype control Ab. The results
showed that IFN- production to M. leprae in the presence
of IL-18 was inhibited by 50–80% by anti-IL-12 (Fig. 4), suggesting that IL-18 and IL-12
collaborate in the immune response against M. leprae.
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production after stimulation with M.
leprae. PBMCs from T-Lep patients were stimulated with Ag in the
presence or absence of anti-IL12 Ab, anti-IL-18 Ab, or
anti-IL-12 plus anti-IL-18 Abs. The results showed that both
neutralizing mAbs considerably inhibited M. leprae-induced
IFN- production, indicating that the two cytokines are involved in
the immune response against M. leprae (Fig. 5). Moreover, when IL-12 and IL-18 were
added together to PBMCs from T-Lep patients in culture with M.
leprae, a significant increase in the IFN- production was
observed (Fig. 6). These data suggest
that IL-12 and IL-18 act together to enhance IFN- production in
response to M. leprae.
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production to M.
leprae
To identify the population(s) of cells that respond to IL-18,
depletion of CD3+ cells or CD56+ cells were
performed from PBMCs of T-Lep patients. After the depletion, the cells
were stimulated with M. leprae in the presence or absence of
IL-18 for 24 h. M. leprae-stimulation of the
CD3-depleted population induced high levels of IFN- in response to
IL-18 (Fig. 7). In contrast, M.
leprae-stimulation of CD56-depleted cells did not induce
detectable levels of IFN- in response to IL-18 (Fig. 7). These
results suggest that NK cells were involved in the M.
leprae-induced IFN- production in response to IL-18 after
24 h of stimulation. To further confirm the role of NK cells in
the observed response, additional experiments using triple-color flow
cytometry were performed with PBMCs from T-Lep patients. PBMCs were
stimulated with M. leprae in the presence or absence of
IL-18. After 24 h of incubation, surface staining for CD3 and CD56
and intracellular staining for IFN- was performed. As shown in this
representative donor, 20% of CD56+ cells were positive for
intracellular IFN- (Fig. 8A, lower panel),
whereas <8% of the CD56- cells produced IFN- (Fig. 8A, lower panel), with no increase in the number
of CD3+ cells producing IFN- (data not shown). These
data confirmed that IL-18 induces an early IFN- response by NK
cells.
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production to M.
leprae
In an attempt to determine whether IL-18 induces an IFN-
response in M. leprae-stimulated T cells, Ag-stimulated
PBMCs from T-Lep patients were cultured in the presence or absence of
IL-18 for 5 days. Triple-color flow cytometry for CD3, CD56, and
intracellular IFN- was then performed. Fig. 8B
illustrates the results from one representative donor of five. After 5
days of Ag-stimulation, IL-18 induced a clear increase in the
percentage of IFN--producing cells bearing the CD3 marker (27%),
whereas no changes in the levels of IFN- produced by the
CD3- population were detected (<7%; Fig. 8B).
Moreover, when we analyzed the CD56+ population that
responded to the Ag in the presence or absence of IL-18, our results
showed that 10% of CD56+ cells produced
IFN-+ (CD56+ IFN-+) upon
stimulation with M. leprae, but no change was detected in
response to M. leprae plus IL-18 (Fig. 8B). As
CD56+ cells include two populations, NK cells and T cells,
which can be differentiated by the expression of CD3, our results
showed that, after 5 days of Ag-stimulation, IL-18 induced no change in
the percentage of NK cells producing IFN-, but clearly increased the
percentage of IFN--producing cells bearing the CD3 marker.
To confirm the effect of IL-18 on Ag-stimulated T cells, we stimulated
T cell lines with Ag, in the presence or absence of IL-18, for 12
h, and IFN- levels were measured. In these experiments, T
cells and double-negative 
ß T cells were used. Stimulation of the
T cell clone HF.2 with increasing concentrations of IL-18 in the
presence of IPP induced high levels of IFN- as compared with
stimulation in the absence of Ag (Fig. 9A). Moreover, stimulation of
double-negative cells from the T cell line LDN4 with IL-18 in the
presence of lipoarabinomannan induced higher levels of IFN- as
compared with stimulation with the cytokine in the absence of Ag (Fig. 9B). These data show that IL-18 considerably enhanced the
IFN- production from Ag-stimulated T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(but not IL-4)
production in T-Lep patients as compared with L-Lep patients. A
combined effect of IL-12 and IL-18 in promoting M.
leprae-specific type 1 responses was also demonstrated. Finally,
we characterized the cell populations involved in the early and late
response to M. leprae in the presence of exogenous IL-18,
demonstrating an important role of NK cells in the early response (1
day), followed by a delayed (5 day) response by T cells. Taken
together, these data indicate that IL-18 is part of the matrix of
proinflammatory cytokines produced by monocytes, which contributes to
CMI against infection in humans.
IL-18 mRNA was induced by M. leprae, although protein
expression was low. However, IL-18 was locally detected in leprosy
lesions from T-Lep patients, the group of individuals with greater CMI
to M. leprae. The local production of IL-18 in tuberculoid
lesions, along with the production of IL-12 (3), would be expected to
facilitate the type 1 cytokine response necessary for CMI against
infection by the pathogen. Conversely, low levels of IL-18 mRNA were
found in L-Lep patients, the group of individuals with weak CMI to
M. leprae. The low levels of IL-18, along with the low
levels of IL-12 in these patients, likely contribute to the weak
IFN- response in this group. Recent studies have suggested that
IL-18 play a role in the host defense against a range of microbial
pathogens (16, 17, 18, 21, 34). Regarding bacterial infection, it was
suggested that IL-18 might participate in the clearance of
Yersinia infection (18) and in the protection against
experimental M. leprae infection in mice (19). In humans,
several strains of nonpathogenic Lactobacillus and
pathogenic Streptococcus were shown to induce IL-18
production in PBMCs (20). Our present data suggest that the local
production of IL-18 in leprosy appears to have an integral role in
determining the nature of the host immune response.
Previously we have demonstrated that IFN- production in response to
microbial Ag is enhanced by IL-12 and IL-15 (35). Our present data also
indicated that IL-18 augmented IFN- production in response to
M. leprae. To elucidate the mechanism for IFN- production
by IL-18, we studied the cell populations involved in this response. We
found that upon stimulation with M. leprae, IL-18 induced an
early IFN- response from NK cells, mediators of innate immunity, and
a later IFN- response from T cells, mediators of adaptive immunity.
The effect of IL-18 on Ag-stimulated T cells was further confirmed
using T cell lines. For this purpose we stimulated ß and T
cell lines with specific Ags. The innate immune response pertains to
those cells preprogrammed to respond to non-self stimuli, in contrast
to the adaptive response that involves the selection and expansion of
immune cells, such as T and B cells, with the development of
immunologic memory (4). The production of IL-18 from cells of both the
innate and adaptive immune responses would allow these two arms of the
immune system to act in concert, to cooperate in the induction of
IFN- release to generate CMI against the pathogen.
Specific unresponsiveness of T cells to M. leprae is the
primary immunologic characteristic of L-Lep patients. It has been shown
that rIL-12 augmented M. leprae-specific T cell
proliferation in L-Lep patients, increasing T cell IFN- production
(23). Because IL-18 was demonstrated to share some of its biological
activities with IL-12 (12, 14), and given the dependence of M.
leprae-induced T cell proliferation on IL-12, we investigated
whether IL-18 could enhance Ag-induced T cell responses in L-Lep
patients. However, while IL-18 increased the type 1 cytokine response
from T-Lep patients, L-Lep patients showed no response to IL-18,
demonstrating that, in marked contrast with the effect of IL-12 on
M. leprae-specific T cell responses, L-Lep patients are
unresponsive to IL-18 stimulation. The mechanism for this
unresponsiveness requires further investigation. However, it was
recently demonstrated that IL-12 and IL-18 act differentially to
activate IFN- gene transcription in primary human CD4+ T
cells (36). It was shown that while both AP-1 and STAT4 are required
for IL-12-dependent IFN- promoter activation, IL-18 causes direct
promoter activation via AP-1. It has been suggested that this
differential activation of the IFN- promoter gives further insights
into molecular pathways governing Th1 T cell development and
differentiation (36). Thus, the mechanism whereby IL-18 enhances
M. leprae-specific IFN- production might be through
direct activation of AP-1 site in the IFN- promoter (36).
Recent studies have demonstrated that IL-18 and IL-12 synergistically
induced the production of IFN- by NK, T, and B cells (10, 11, 13, 15). In addition, exogenously administrated IL-12 and IL-18 acted
synergistically to protect mice against cryptococcal infection (16).
These findings may suggest that the two IFN--inducing cytokines
synthesized in vivo collaborate and strengthen their protective
activities against infection by inducing the production of IFN-.
Using double knockout mice lacking both IL-18 and IL-12, these
cytokines have been shown to induce NK activity as well as in vivo Th1
development (37). Moreover, Robinson et al. showed marked synergy
between IL-18 and IL-12 in inducing IFN- production from
differentiating and committed Th1 cells from mice, suggesting that both
IL-12 and IL-18 are required for expression of the Th1 phenotype (38).
Our studies demonstrated that IL-18 and IL-12 collaborate in modulating
Ag-specific responses by inducing IFN-.
In conclusion, we investigated the role of IL-18 in the immune response to M. leprae in the hope of developing a model to explain the profound cytokine differences observed in the polar manifestation of leprosy. The local production of the type 1 cytokine pattern in T-Lep patients would promote CMI responses to M. leprae, allowing them to control the growth of the bacteria and to respond to the infection. On the contrary, the absence of type 1 cytokine responses in L-Lep patients would contribute to the weak or absent CMI responses to the pathogen, resulting in progressive infection. We have demonstrated that IL-18 can up-regulate NK and T cell functions against M. leprae, providing evidence that IL-18 is an important mediator in generation of the type 1 cytokine response in human infectious disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert L. Modlin, Division of Dermatology, 52–121 CHS, University of California, Los Angeles, School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address:
3 Abbreviations used in this paper: CMI, cell-mediated immunity; T-Lep, tuberculoid leprosy; L-Lep, lepromatous leprosy; RT, room temperature; IPP, isopentenyl pyrophosphate; HuIGIF, human IFN--inducing factor.
Received for publication April 27, 1998. Accepted for publication February 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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