| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-mediated
protective immune responses. We tested the effects of rIL-12
administration to Mycobacterium avium-infected mice and
found a limited ability to induce protection against the infection;
this ability varied according to the mycobacterial strain
studied. IL-12 accelerated the expression and production of
IFN- in both immunocompetent and immunodeficient SCID or
CD4-depleted mice. Evidence of NK cell activation was found as well as
an enhancement of the ability to adoptively transfer resistance with T
cell-enriched spleen cell populations and an increase in inflammatory
cell recruitment in the liver. The protective ability of IL-12 was
dependent upon the endogenous production of IFN- as evaluated by the
use of specific neutralizing Abs or IFN- gene-disrupted mice. IL-12
potentiated the protective immunity conferred by a subunit vaccine
containing M. avium culture filtrate proteins and
dimethyl dioctadecyl ammonium chloride as an adjuvant. Thus, we show
limited immunotherapeutic benefits from IL-12 administration in
M. avium infections and promising results in its use as
a coadjuvant in vaccine design. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dominated
immune responses to microbial pathogens. Studies using neutralizing Abs
or gene-disrupted animals have underscored the almost absolute
requirement for IL-12 in the generation of protective Th1 immunity
(1, 2, 3, 4, 5, 6, 7, 8, 9). Likewise, the exogenous administration of recombinant cytokine
led to protection against different microbes (4, 6, 10, 11, 12, 13, 14, 15, 16, 17), including
mycobacteria (1, 18, 19, 20). In addition, other studies have described the
adjuvanticity of IL-12 in vaccines against parasites such as
Leishmania, where it increased protection (21), and
Schistosoma, where it decreased pathology (22). On the other
hand, the genetic basis of susceptibility to infection was related in
some cases to defective IL-12 production (19). Human patients have also
been identified that are particularly prone to mycobacterial infections
due to defective IL-12 production or signaling (23).
Mycobacterium avium is a facultative intracellular pathogen
that frequently infects immunocompromised individuals. Several reports
indicate that M. avium infection is one of the most common
causes of systemic bacterial infection in AIDS patients in the United
States and western Europe (24). Most AIDS patients develop disseminated
M. avium infections when their circulating CD4+
T cell numbers are <100/mm3 (25), suggesting an important
role for this T cell subset in the protective immune response against
this pathogen. Disseminated M. avium infections have also
been described in human patients deficient in the IFN-R (26) in
addition to those defective in IL-12 secretion or responsiveness
to IL-12.
BALB/c mice are naturally susceptible to M. avium
infections. However, the growth of certain M. avium strains
(such as strain 2447) can be arrested after the emergence of protective
CD4+ T cells. These cells secrete IFN-, which induces
bacteriostasis in infected macrophages (27). One of the molecules
involved in the modulation of this protective immune response is IL-12.
In this murine model of infection, endogenously produced IL-12 acts in
an early phase of the infection by M. avium 2447, inducing
protective T cell-independent mechanisms and, later on, promoting the
differentiation of IFN--producing CD4+ T cells (28).
In this study, we evaluated the immunomodulatory effects of the administration of murine rIL-12 (rmIL-12)3 on the course of M. avium infection in BALB/c mice. The effects of the administration of rmIL-12 were analyzed with respect to its role in both the emergence of protective CD4+ T cells and in T cell-independent protective mechanisms. Also, the usefulness of IL-12 as an adjuvant in subunit vaccines against M. avium was assessed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mycobacterial growth media were purchased from Difco (Detroit,
MI). RPMI 1640 cell culture medium and FCS were obtained from Life
Technologies (Paisley, Scotland). Tween 80 and IFA were
purchased from Sigma (St. Louis, MO). Anti-CD4 mAbs were obtained from
the hybridoma GK1.5 (TIB 207 cell line; American Type Culture
Collection (ATCC), Manassas, VA). The IFN--neutralizing IgG1 was
obtained from the hybridoma XMG1.2 (DNAX, Palo Alto, CA), and an
irrelevant rat IgG1 against ß-galactosidase was purified from
hybridoma GL113 (DNAX). Hybridomas were grown in ascites in Harlan
Sprague-Dawley nude mice primed with IFA. Abs were purified by
affinity chromatography using a protein G-agarose column (Life
Technologies). rmIL-12 was a kind gift of the Genetics Institute
(Cambridge, MA) and had a specific activity of 3.7 x
106 U/mg.
Mice
Specific pathogen-free BALB/c female mice were purchased from
the Gulbenkian Institute for Science (Oeiras, Portugal). Outbred Harlan
Sprague-Dawley nude mice were purchased from the Gulbenkian
Institute for Science and used to raise ascites from hybridomas. C.B-17
SCID female mice were purchased from Bommice (Ry, Denmark), and
IFN-gamma gene-disrupted (IFN--/-) mice were
generated by Dalton et al. (29). These mice were kept in sterile
housing conditions in cages provided with high-efficiency particulate
air filter-bearing caps. All mice were used at 6–8 wk of age.
In some experiments, CD4+ T cell-depleted BALB/c mice were obtained by thymectomy and anti-CD4 mAb administration as described previously (27).
Bacterial infections
M. avium strains 25291 (from ATCC), 2447 (an AIDS isolate obtained from Dr. F. Portaels, Institute of Tropical Medicine, Antwerp, Belgium), and 1983 (a low virulence isolate from an HIV-negative patient) were grown in Middlebrook 7H9 medium containing 0.04% Tween 80 at 37°C until the mid-log phase of growth. Bacteria were harvested by centrifugation and resuspended in a small volume of saline containing 0.04% Tween 80. The suspension was briefly sonicated with a Branson sonifier (Danbury, CT) for 15 s at 50 W to disrupt bacterial clumps, diluted, and frozen in aliquots at -70°C until use.
Mice were infected i.v. with 108 CFU of M. avium 1983 or 106 CFU of M. avium 2447 or 25291 through a lateral tail vein. Mice were sacrificed by cervical dislocation at different timepoints, and organs were collected, homogenized, serially diluted in a 0.04% Tween 80 solution in distilled water, and plated onto Middlebrook 7H10 agar medium. The plates were incubated for 2 wk at 37°C, and the number of CFU were counted. Data are expressed as the log10 value of the mean number of CFU recovered per organ ± SD of the mean (n = four or five animals). Differences were analyzed by the Student t test using unpaired data.
Cytokine assays
Serum was obtained from blood collected from sacrificed mice
after allowing clot formation at 37°C for 30 min followed by an
incubation at 4°C for 2 h to allow clot retraction. After
centrifugation (10,000 rpm for 20 min at 4°C), serum was collected
and frozen at -70°C until use. IFN- levels in individual sera
were detected and quantified by a two-site sandwich ELISA using
anti-IFN--specific affinity-purified mAbs (R4-6A2 as capture and
biotinylated AN-18 as detecting) and a standard curve generated with
known amounts of rmIFN- (Genzyme, Cambridge, CA). The sensitivity of
the assay was 80 pg/ml.
Treatment of mice with mAbs and cytokines
To maintain CD4+ T cell depletion in thymectomized
animals, 0.2 mg of anti-CD4 mAb (GK1.5) per animal was administered
i.p. every 10 days during the experimental infection. For IFN-
neutralization, 2 mg of XMG1.2 per animal was injected i.p. every 2 wk
from the beginning of infection. Control mice received the same amount
of purified anti-ß-galactosidase mAb of the same isotype (GL113
mAb).
Treatment with rmIL-12 was performed by administrating 0.4 µg of this cytokine per animal i.p. on every other day during 1 mo from the beginning of the infection or during 3 wk starting on day 21 of infection.
Adoptive transfer of spleen cells
Spleens from BALB/c mice that had been infected for 3 or 6 wk with M. avium strain 2447 or left uninfected and treated with rmIL-12 or left untreated were aseptically collected and gently teased in RPMI 1640 medium containing 2% FCS (AT medium). Splenic cells were pooled (n = 4), washed, and resuspended in hemolytic buffer (155 mM NH4Cl, 10 mM KHCO3, pH 7.2) for 5–10 min at room temperature. After two washes in AT medium, cells were resuspended in the same medium plus an equal volume of J11d.2 (anti-CD24) and MK-D6 (anti-class II MHC) hybridoma supernatants (total volume of 30 ml) and incubated with rabbit complement (1:30 dilution) for 45 min at 37°C. Clumps of cells and cell debris were discarded, and cells were washed twice in AT medium. Cells were resuspended in AT medium, added to a nylon column containing 1.2 g of wool in a 10-ml syringe barrel, and incubated for 2 h at 37°C. Nonadherent cells were collected by washing the column with 20 ml of warm AT medium. This fraction was washed twice and injected i.v. in 0.5 ml of the same medium to recipient mice that had been irradiated with a Cs source (500 rad/mouse) 24 h earlier. One spleen equivalent was injected per recipient mouse. After 2 h, recipient mice were challenged i.v. with 106 CFU of M. avium 2447. Mice were killed 30 days later, and viable counts were done on the spleens and livers as described above. The protection achieved was calculated by subtracting the geometric mean of CFU in the organs of mice receiving immune spleen cells from that in mice receiving control spleen cells. The percentage of CD4+ T cells in the final cell suspension varied from 36 to 51%.
Flow cytometry
Single-cell suspensions from the spleens of treated or control
infected SCID mice were prepared by teasing a portion of the spleen in
medium. For immunofluorescence double staining, 106 cells
were incubated in microtiter plates for 20 min at 4°C with
heat-inactivated rat serum. Cells were washed and subsequently
incubated for 30 min at 4°C with FITC-conjugated rat anti-mouse
CD90.2/Thy-1.2 (Becton Dickinson, Mountain View, CA) or rat
anti-mouse CD11b/Mac1 and R-phycoerythrin (PE)-conjugated
hamster anti-mouse CD3
mAbs (PharMingen, San Diego, CA). Cells
were washed twice with staining medium (PBS containing 0.1% sodium
azide and 5% FCS) and resuspended in staining medium containing
propidium iodide to identify and exclude dead cells. Flow cytometric
analysis was performed with a FACSort apparatus (Becton Dickinson)
equipped with Lysis II software by acquiring 10,000 events.
To analyze the T cell-enriched splenocytes from uninfected and infected donor mice in the adoptive transfer experiments, 106 cells from the T cell pools were stained with PE-conjugated anti-mouse CD4 and FITC-conjugated anti-mouse CD8 (PharMingen) as described above.
Histology
Portions of the liver or spleen of infected mice were cut from the organs with the aid of a sharp blade, fixed in buffered formaldehyde, and embedded in paraffin. Next, 3-mm-thick sections were cut and stained for acid-fast bacilli by the Ziehl-Neelsen method and either counterstained with hematoxylin or stained with trichrome. Slides were analyzed for differences in the number and size of granuloma structures as well as for the presence or absence of acid-fast rods. Representative fields were photographed with a Nikon microscope (Tokyo, Japan).
RT-PCR
Total RNA from spleen cell suspensions was isolated by
guanidinium thiocyanate/phenol/chloroform purification and
stored at -70°C until further processing. Total mRNA was reverse
transcribed using p(dT)12–18 oligonucleotides (Pharmacia
Biotech, Uppsala, Sweden) as primers and Superscript reverse
transcriptase (Life Technologies) in the presence of 10 U of RNase
inhibitor (Promega, Madison, WI) (30). Amplification of cDNA was
conducted with Taq polymerase (Perkin Elmer-Roche Molecular
Systems, Branchburg, NJ) and specific primers for hypoxanthine
phosphoribosyltransferase (HPRT) or IFN- in a Gene Amp PCR
System 9600 (Perkin Elmer) (31) for 30 cycles. The primers used have
been described previously (30). Care was taken to analyze the product
generated under conditions of linear relation to the concentration of
the cDNA. After standardization for HPRT mRNA, the amplification
products were run in a 1.5% agarose gel, transferred to nitrocellulose
membranes (Hybond N+; Amersham, Buckinghamshire, U.K.), and
hybridized with specific probes labeled with
[
-32P]deoxyCTP. Membranes were exposed, and
photographic plates were read with the aid of a computer-assisted
scanner. Values for the PCR product for IFN- were corrected for the
amount of HPRT on each sample, taking into account the titration of
both the HPRT and IFN- cDNA from internal standards. All samples and
the titrations were run, blotted, and exposed in parallel to the same
plates to ensure a correct comparison of the signals generated. Data
are expressed as the mean pixel value of four or five samples from
separate mice. Values were analyzed using the Student t
test.
Vaccine preparation and immunization studies
M. avium 2447 was grown for 2 wk in Sauton medium supplemented with pyruvate and glucose; culture supernatants were obtained by centrifugation of the bacteria and filtration through 0.45-µm filters. Such culture supernatants were concentrated 100-fold through ultrafiltration using filters with a molecular mass cutoff of 3 kDa. The resulting preparation was then subjected to 80% ammonium sulfate precipitation followed by extensive dialysis against PBS. Therefore, this reagent was named culture filtrate protein from M. avium (CFP). To immunize animals, 45 µg of CFP was injected three times s.c. at the base of the tail in separate locations, at weekly intervals. CFP was administered as a solution or was admixed with 0.4 µg of rmIL-12 and/or 250 µg of dimethyl dioctadecyl ammonium chloride (DDA) (Eastman Kodak, Rochester, NY) as adjuvants. The control mice received either PBS alone, rmIL-12 alone, DDA alone, or rmIL-12 in DDA. At 1 mo after the last immunization, mice were challenged i.v. with 106 CFU of M. avium strain 2447. The mycobacterial loads in the spleen and liver were determined 30 days later as described above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BALB/c mice were infected i.v. with 106 CFU of either
of two strains of M. avium of distinct virulence
(characterized in 31 and treated every other day with rmIL-12
(each dose containing 0.4 µg of IL-12/animal) or PBS i.p. during the
first month of infection. The growth of M. avium was studied
in the spleen and liver of the infected animals for 60 days. The
results showed that rmIL-12 induced statistically significant
protection in the spleens of BALB/c mice infected with strains 25291
and 2447 but had no effect on the mycobacterial growth in the liver
(Fig. 1
A). The protection
afforded in the spleen against the most virulent strain 25291 was
minimal when compared with the overall growth of the mycobacteria,
whereas a 0.9 log10 decrease in mycobacterial numbers was
detected in the spleens of rmIL-12-treated mice infected with the low
virulence strain 2447 as compared with the PBS-treated control group.
After discontinuation of the treatment, differences in splenic
mycobacterial loads between the two groups of mice decreased slightly
to 0.6 log10, but significantly lower numbers were still
observed in the rmIL-12-treated group as compared with the controls.
Since our results were different from those reported previously by
Kobayashi et al. (19), who described the ability to induce the killing
of M. avium, we chose to study M. avium strain
1983, which has virulence characteristics similar to the strain used in
that study. Furthermore, we adapted the therapeutic regimen to the one
used by those authors (i.e., 3 wk of cytokine administration starting
on day 21 of infection). In addition, we compared the effects of such a
regimen on the growth of strain 2447. As shown in Fig. 1B,
this second IL-12 treatment protocol was not effective in reducing the
mycobacterial load during infection by strain 2447. However, we could
detect a 0.8 log10 difference in the splenic loads and a
very minor although statistically significant decrease in hepatic loads
of 0.3 log10 during infection by the low virulence strain
1983. Furthermore, we found killing of mycobacteria in the spleens of
mice infected with this latter strain; this killing took place after
the administration of the cytokine.
|
. Analysis was performed by RT-PCR of IFN- splenic expression
in BALB/c mice treated i.p. with rmIL-12 (0.4 µg/animal every other
day) or PBS and infected i.v. for 30 days with 106 CFU of
M. avium strains 25291 and 2447. As shown in Fig. 2, treatment with rmIL-12 caused a minor
increase in mRNA levels for IFN-; however, this increase was not
statistically significant. Since the timepoint chosen represents the
peak of expression for IFN- during M. avium infections
(25, 31), and as the treatment with rmIL-12 might be accelerating its
expression rather than increasing the peak expression of the cytokine,
we selected strain 2447 and studied earlier timepoints of infection.
Mice were infected as described, and half were treated with rmIL-12 as
before. As shown in Fig. 3A,
protection was induced by rmIL-12 therapy in both the spleen and liver
and was already apparent on day 10 of infection. This early protection
was associated with higher expression of IFN- mRNA on day 10
in rmIL-12-treated animals as compared with the controls (Fig. 3, B and C). However, such differences disappeared
on day 20. The levels of immunoreactive IFN- present in the sera of
the infected animals correlated well with the RT-PCR studies, in that
rmIL-12 treatment induced high levels of IFN- on day 10 that
decreased by day 20 of infection, such amounts of the cytokine always
being higher in the rmIL-12-treated group as compared with the controls
(Fig. 3D).
|
|
The early enhanced production of IFN- could be due either to an
acceleration of the T cell responses or to the boosting of innate
immunity, namely of those mechanisms involving cells of the NK cell
lineage. To evaluate the effects of rmIL-12 administration on T
cell-independent immunity, SCID mice were infected i.v. with
106 CFU of M. avium 2447 for 1 mo; 0.4 µg of
rmIL-12 was administered every other day from the beginning of the
infection to one group of animals. Fig. 4A shows the mycobacterial
load in the spleen and liver of the animals infected for 30 days. The
administration of rmIL-12 potentiated the protective response in the
spleens of SCID mice. However, the magnitude of the reduction in
M. avium proliferation induced by rmIL-12 treatment was
smaller than the one previously observed with immunocompetent BALB/c
mice. The sera of rmIL-12-treated SCID mice had detectable levels of
immunoreactive IFN-; IFN- was not detected in the control mice
(Fig. 4B).
|
C).
This cytokine also induced an increase in the granularity of the
CD3-Thy1.2+ and
CD3-Mac1+ cell populations (Fig. 4, D and E, respectively), which suggests an
activation of these cell populations. Similar studies were performed on thymectomized BALB/c mice treated with anti-CD4 mAbs. Such mice, when given rmIL-12 in a protocol similar to that used in the SCID mice, showed reductions in splenic M. avium loads in two independent experiments of 0.3 and 0.7 log10 at day 15 and of 0.4 and 0.7 log10 at day 30 as compared with CD4-depleted mice that did not receive the cytokine; this finding indicates that, in this model, the effects of rmIL-12 take place during the first 2 wk of treatment. Furthermore, at day 15 of infection, CD4-depleted mice had the same splenic mycobacterial loads as control thymectomized mice in either the cytokine-treated groups (5.22 ± 0.02 log10 CFU vs 5.24 ± 0.08 log10 CFU) or the groups that did not receive rmIL-12 (5.88 ± 0.02 log10 CFU vs 5.99 ± 0.14 log10 CFU), showing that the early protection at day 15 in this experiment was independent of CD4+ T cells. Later on, however, the protection induced by IL-12 was higher in control compared with CD4-depleted animals. Thus, at day 30 of infection, the protection in the spleens of control mice was 1.1 log10, whereas protection in CD4-depleted animals was 0.7 log10.
Evaluation of the effects of rmIL-12 treatment on T cell-dependent immunity
To test the effects of the rmIL-12 treatment on the T cell
populations, we studied the protective activity of T cell-enriched
spleen cells from infected animals treated or untreated with the
cytokine using a passive cell-transfer assay. A total of 16 donor mice
were infected i.v. with 106 CFU of M. avium 2447
for 3 or 6 wk. Half of the animals were given rmIL-12 (0.4 µg/animal
every other day for the entire period), and the other half were treated
with PBS. Spleen cells from these immune animals as well as spleen
cells from nonimmune controls were collected, enriched for T cells by
depletion of Ia+ cells and nylon wool-adherent cells, and
infused into irradiated (500 rad) recipient animals. The latter were
then challenged with 106 CFU of the same M.
avium strain, and mycobacterial growth was evaluated 30 days
later. We had already determined that the protection afforded by these
cells can be abrogated by in vitro lysis of CD4+ T cells by
treatment with specific Abs and complement (data not shown). Fig. 5 shows that the injection of rmIL-12 in
M. avium-infected donor mice accelerated the emergence of a
population of T cells that were able to adoptively transfer protection
to sublethally irradiated recipient mice.
|
To test whether the protective effect of rmIL-12 in
immunocompetent animals was dependent upon IFN-, we compared the
effects of rmIL-12 treatment on the growth of M. avium in
BALB/c mice given neutralizing Abs vs respective controls or in IFN-
gene-disrupted animals (IFN--/-) and normal
heterozygous controls (IFN-+/-). Mice were infected
i.v. with 106 CFU of M. avium 2447 for 1 mo and
treated every other day with either PBS or 0.4 µg of rmIL-12/animal.
Fig. 6 shows the mycobacterial loads in
spleens and livers of mice infected for 30 days. The protective effects
of rmIL-12 in the spleen were dependent upon the endogenous production
of IFN-. Such effects were particularly evident with the
IFN--/- mice, but similar results were obtained when
immunocompetent BALB/c mice were used and specific neutralizing Abs
were given to deplete IFN-. Fig. 7
shows the histologic analysis of the livers of these animals. Untreated
IFN--/- mice had a marked decrease in granuloma
formation during infection as compared with control heterozygous mice.
Treatment of mice with rmIL-12 increased granuloma formation both in
control and IFN--deficient animals. Analysis of the spleens showed
invasion of the white pulp by macrophages during infection. Although
granulomas were less easily discernible within the lymphoid mass, their
number was enhanced by the infusion of rmIL-12 in both immunocompetent
and IFN--/- mice (data not shown).
|
|
To evaluate the possible usefulness of IL-12 in the design of
antimycobacterial vaccines, we selected secreted proteins from M.
avium as the Ag source. This choice was based on the previous work
of several groups that described the effective use of such preparations
in the induction of protective immunity against Mycobacterium
tuberculosis (32). CFPs were injected either alone or in
combination with DDA as an adjuvant (33). rmIL-12 was administered
together with CFP or CFP plus DDA to subgroups of animals. Controls for
the nonspecific, inflammatory-dependent induction of resistance to
infection included the administration of rmIL-12 and DDA, both alone or
in combination, as well as the vehicle, PBS. We chose to administer the
cytokine with every dose of the vaccine, since preliminary results
showed no benefit of a single dose during the first immunization (Ref.
33 and our unpublished observations). As shown in Fig. 8, significant resistance was conferred
by the adjuvants alone in this experiment when the challenge took place
1 mo after the last immunization. This was particularly evident in the
liver. However, all three preparations containing the Ag, CFP, admixed
with DDA, rmIL-12, or both conferred significantly higher protection in
the liver than any of the other preparations lacking Ag or the CFP
treatment alone, without any adjuvant. In the spleen, in contrast, high
levels of protection (p < 0.01 toward all
other groups) leading to 10-fold lower numbers of mycobacteria were
only obtained when CFP was administered in combination with both DDA
and rmIL-12.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-activated macrophages (Refs. 34 and 35 and our unpublished
observations). Therefore, further activation of those pathways is
without success in the control of such infections. The effects of
rIL-12 administration to mice infected with M. avium have
already been studied by Kobayashi et al. (19). They found that IL-12
administered in a regimen very similar to the one used here for strain
1983 (a strain chosen to have a virulence similar to that of the strain
used in their study (36)) led to a killing of the mycobacteria in the
spleens of infected animals. We corroborate their observations, but
stress that the efficacy of the IL-12 treatment is clearly dependent
upon the characteristics of the particular strain of M.
avium studied. This information may turn out to be of importance
if IL-12 is considered for human immunotherapy. We found no clinically
apparent toxic effects of prolonged IL-12 therapy, but histologic
analysis of the infected livers showed a marked increase in the
granulomatous response. This enhancement was partially independent of
the infection itself, since we observed marked cellular infiltration
and granuloma formation in the livers of uninfected mice treated with
similar regimens of rmIL-12 (our unpublished observations). Part
of the toxicity of IL-12 was reported to be dependent upon the
triggering of TNF- production (37). Since TNF- is also involved
in resistance to M. avium infection (27), the neutralization
of this cytokine during IL-12 therapy to decrease the toxicity of
therapy would not be warranted. The limited effects of rmIL-12 therapy
on the overall growth or killing of mycobacteria have also been
documented for other mycobacterial species. In one report, the
protection afforded by rmIL-12 against a tuberculous challenge in
BALB/c mice was also limited to one log10; however, in that
model, such improvement of the control of the infection prolonged the
survival of the mice (20). Cooper et al. (1, 18) have also described
low levels of protection in an i.v. challenge model of murine
tuberculosis. Here, we found that the less virulent the strain of
M. avium, the bigger the protection conferred by rmIL-12
therapy. Finally, a recent report that appeared after our initial
submission of this work shows similar levels of protection after IL-12
therapy of SCID mice infected with M. avium (38). In that
study, IL-12 was shown to potentiate the efficacy of chemotherapy.
We used strain 2447 to dissect the basis of the protective effect
afforded by IL-12. Such protection was dependent both on the induction
of innate mechanisms involving the activation of macrophages and NK
cells and on the acceleration of the T cell response. Both cell types
were responsible for the enhanced early production of IFN- that was
required for the decrease in mycobacterial proliferation. This was
shown using SCID mice or CD4-depleted mice to evidence the innate
immunity mechanisms and by performing an adoptive transfer of
protection to highlight the role of T cells. Also, the protective
effects of IL-12 therapy were higher in immunocompetent than in T
cell-deficient animals and were only expressed against strain 2447 when
the cytokine was given at a time when T cell-mediated immunity was
emerging, rather than when it was already present (i.e., at wk 3 of
infection) (Fig. 1, A vs B). However, the
relative importance of either of the protective mechanisms triggered by
IL-12 in an immunocompetent mouse was not clear.
In accordance with other studies (20), we found that the protective
effect of IL-12 was dependent upon the endogenous production of IFN-
and was associated with an increased development of granulomatous
inflammation in the liver. In the absence of IFN-, IL-12 was also
able to increase the recruitment of inflammatory cells despite its
inability to protect from infection. A similar dependence of the
effects of IL-12 on the endogenous production of IFN- has also
been found in other infectious models (10, 11, 12, 14, 16, 17).
More promising to our point of view than the immunotherapeutic potential of rmIL-12 were the findings of the potentiation of the protective immune responses elicited by CFPs by IL-12 coadministration. Although the results are clearly preliminary and more extensive studies should be performed to assess the longevity of the protective immunity generated, it is interesting to note that protection of one log10 or more was found in both the target organs studied only when rmIL-12 was included in the vaccine. It will be important to subject the animals to a more prolonged resting period between immunization and challenge to eliminate the nonspecific protection induced by preparations devoid of Ag, which were studied here to control for the specificity of the vaccine. The limited albeit significant protection afforded by the adjuvant preparations alone in this experiment was most likely due to the accumulation of inflammatory cells at the sites of infection induced by the presence of the adjuvants given only 1 mo before the infection. These cells may be producing cytokines that play a modest protective role on the subsequent infection. Nevertheless, despite the existence of such nonspecific protection, the Ag-specific protection was clearly above the former; most of all, it could only be found in the spleen if rmIL-12 was included in the vaccine. It was curious to note that although the protection afforded by immunotherapy during infection was particularly evident in the spleen, the adjuvant effects of IL-12 were more marked in the liver of the animals. The superiority of the immunotherapy in the spleen can be explained by the later emergence of protection in this organ during infection as compared with the liver. Therefore, treatment with rmIL-12 accelerates protective mechanisms, so that protection appears sooner in the spleen, while protection in the liver may not be further accelerated. This easier recruitment of T cell-mediated protection to the liver as compared with the spleen can also be seen with the vaccinated animals where suboptimal regimens (CFP with only one of the adjuvants) were already effective, whereas the combination of the three components of the vaccine was necessary to boost the protective efficacy of the immunization to act in the spleen.
In summary, we illustrate the variable protection afforded by
immunotherapy with rmIL-12 in murine models of M. avium
infection; this protection depended upon the virulence of the
mycobacterial strains being tested. The protection was dependent
upon the endogenous production of IFN- produced by cells from the NK
cell lineage or from protective T cells that emerged more rapidly when
the cytokine was provided exogenously. The inability to boost the peak
of maximum IFN- response was probably the reason for the limited
protection afforded by the cytokine. However, the use of rmIL-12 as a
coadjuvant in vaccine preparations was a most promising one.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Rui Appelberg, Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre 823, 4150 Porto, Portugal. E-mail address:
3 Abbreviations used in this paper: rmIL-12, murine rIL-12; PE, phycoerythrin; HPRT, hypoxanthine phosphoribosyltransferase; CFP, culture filtrate protein from M. avium; DDA, dimethyl dioctadecyl ammonium chloride.
Received for publication April 2, 1998. Accepted for publication July 13, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
interferon production in mouse typhoid. Infect. Immun. 64:189.[Abstract]
. J. Immunol. 155:785.[Abstract]
and interferon. Infect. Immun. 64:2381.[Abstract]
-dependent protection against malaria. Proc. Natl. Acad. Sci. USA 91:10700.-receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335:1941. interferon and tumor necrosis factor- during the T cell-independent and -dependent phases of Mycobacterium avium infection. Infect. Immun. 62:3962. genes. Science 259:1739. interferon. Infect. Immun. 57:239. interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria. Infect. Immun. 50:1.This article has been cited by other articles:
|
|
 |
|
  C. G. Feng, M. Kaviratne, A. G. Rothfuchs, A. Cheever, S. Hieny, H. A. Young, T. A. Wynn, and A. Sher NK Cell-Derived IFN-{gamma} Differentially Regulates Innate Resistance and Neutrophil Response in T Cell-Deficient Hosts Infected with Mycobacterium tuberculosis J. Immunol., November 15, 2006; 177(10): 7086 - 7093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Florido, J. E. Pearl, A. Solache, M. Borges, L. Haynes, A. M. Cooper, and R. Appelberg Gamma Interferon-Induced T-Cell Loss in Virulent Mycobacterium avium Infection Infect. Immun., June 1, 2005; 73(6): 3577 - 3586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Nolt and J. L. Flynn Interleukin-12 Therapy Reduces the Number of Immune Cells and Pathology in Lungs of Mice Infected with Mycobacterium tuberculosis Infect. Immun., May 1, 2004; 72(5): 2976 - 2988. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Leong, J. C. C. Chang, R. Ong, G. Dawes, W. P. C. Stemmer, and J. Punnonen Optimized expression and specific activity of IL-12 by directed molecular evolution PNAS, February 4, 2003; 100(3): 1163 - 1168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Silva, T. F. Pais, and R. Appelberg Blocking the Receptor for IL-10 Improves Antimycobacterial Chemotherapy and Vaccination J. Immunol., August 1, 2001; 167(3): 1535 - 1541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Silva and R. Appelberg Blocking the Receptor for Interleukin 10 Protects Mice from Lethal Listeriosis Antimicrob. Agents Chemother., April 1, 2001; 45(4): 1312 - 1314. [Abstract] [Full Text]
|
|
|
|
|
|
T. F. Pais, J. F. Cunha, and R. Appelberg Antigen Specificity of T-Cell Response to Mycobacterium avium Infection in Mice Infect. Immun., August 1, 2000; 68(8): 4805 - 4810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Pedrosa, B. M. Saunders, R. Appelberg, I. M. Orme, M. T. Silva, and A. M. Cooper Neutrophils Play a Protective Nonphagocytic Role in Systemic Mycobacterium tuberculosis Infection of Mice Infect. Immun., February 1, 2000; 68(2): 577 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Appelberg, I. S. Leal, T. F. Pais, J. Pedrosa, and M. Florido Differences in Resistance of C57BL/6 and C57BL/10 Mice to Infection by Mycobacterium avium Are Independent of Gamma Interferon Infect. Immun., January 1, 2000; 68(1): 19 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. S. Leal, B. Smedegard, P. Andersen, and R. Appelberg Interleukin-6 and Interleukin-12 Participate in Induction of a Type 1 Protective T-Cell Response during Vaccination with a Tuberculosis Subunit Vaccine Infect. Immun., November 1, 1999; 67(11): 5747 - 5754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Florido, A. S. Goncalves, R. A. Silva, S. Ehlers, A. M. Cooper, and R. Appelberg Resistance of Virulent Mycobacterium avium to Gamma Interferon-Mediated Antimicrobial Activity Suggests Additional Signals for Induction of Mycobacteriostasis Infect. Immun., July 1, 1999; 67(7): 3610 - 3618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Gomes, M. Florido, T. F. Pais, and R. Appelberg Improved Clearance of Mycobacterium avium Upon Disruption of the Inducible Nitric Oxide Synthase Gene J. Immunol., June 1, 1999; 162(11): 6734 - 6739. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
