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*
Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal; and
Department of Pathology, The Portuguese Cancer Institute-Porto Regional Centre, Porto, Portugal
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production.
Administration of this mAb later in infection had no effect on its
course, but improved the effectiveness of chemotherapy when the latter
was started in a chronic phase of infection. Also, the anti-IL-10R
mAb acted as an adjuvant in the induction of protective immunity upon
vaccination with a mycobacterial subunit
preparation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemotherapy of infections by nontuberculous mycobacteria such as Mycobacterium avium complex (MAC) is also difficult due to the low susceptibility of this opportunistic pathogen to most commonly used antitubercle compounds (3). Therapy resorts to a combination of several drugs and, as in tuberculosis, is very lengthy. Long-term treatment with macrolides such as clarithromycin, which is highly effective in the treatment of MAC infections (4, 5), leads to the emergence of resistant forms (6, 7, 8), which can be prevented by combining at least two or three drugs (9, 10). However, this in turn can lead to severe side effects (11, 12). Ways to shorten therapeutical courses as well as increasing efficacy of the treatment are thus needed.
Prevention of mycobacterial infections by vaccination is still an area deserving the attention of many due to the low efficacy of BCG. Analysis of BCG vaccination studies has shown a wide variability in the efficacy of this vaccine against tuberculosis, ranging from 0 to 80% according to geographical area of the study, being often useless in developing countries, in which its success would be of major benefit to the populations (13). One of the strategies currently being studied to improve vaccine efficacy is the use of subunit protein preparations. We have previously used the M. avium model to screen for possible immunomodulatory interventions that may increase the protection afforded by the vaccines (14, 15), and found that this model is suitable for such screening similarly to tuberculosis models (16).
IL-10 is a cytokine with pleiotropic activities, being its major effects associated with its antiinflammatory and immunosuppressive properties on hemopoietic cells (17, 18, 19). The increased production of this cytokine has been shown to underlie susceptibility to numerous infections (20, 21, 22, 23). Among mycobacterial infections, IL-10 was shown to promote susceptibility to BCG and MAC (24, 25, 26, 27). The possible mechanisms underlying increased susceptibility to infection may be many different ones, such as modulation of the type and polarization of the immune response; interference with Ag presentation, namely at the level of the dendritic cells; regulation of the immune response; and modulation of the activities on the effector cells, namely deactivation of monocytes/macrophages (17, 18, 19). Regarding the latter effects, IL-10 reduces antimicrobial functions of activated macrophages by decreasing the generation of superoxide anion and NO (28, 29). Inhibition of inflammation relates to the inhibition of the production of proinflammatory cytokines and monokines by activated monocytes/macrophages (30, 31) and to the effects on the immune response due to the down-regulation of the expression of costimulatory molecules and of class II molecules (32, 33, 34, 35). IL-10 may thus also affect the outcome of vaccination namely to subunit vaccines. Recently, a role for IL-10 in the induction of tolerance to soluble Ag was postulated (36). Thus, we reevaluated the participation of IL-10 in the chemotherapy and vaccination of mycobacteriosis using an established mouse model of infection by M. avium. We show that although not explaining susceptibility to infection, IL-10 may be responsible to refractoriness to chemotherapy. Additionally, we postulate a role for inhibitors of IL-10 action as potential adjuvants in subunit vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mycobacterial growth media were purchased from Difco (Sparks,
MD). DMEM, RPMI 1640 medium, HEPES buffer, FCS sodium pyruvate,
penicillin/streptomycin, L-glutamine, and protein G columns
were purchased from Life Technologies (Paisley, U.K.). Tween 80, oleic
acid, 2-ME, potassium bicarbonate, BSA, Con A, IFA, and ethambutol were
purchased from Sigma (St. Louis, MO). Clarithromycin was purchased from
Abbot Laboratories (Amadora, Portugal), and rifabutin (Pharmacia Adria,
Dublin, OH) was supplied by the manufacturers. Mouse rIFN- was
purchased from Genzyme (Cambridge, MA). The IL-10R-blocking IgG1 was
obtained from the hybridoma cell line 1B1.2, a kind gift from K. Moore
(DNAX, Palo Alto, CA) (37). The anti-
-galactosidase
IgG1, an isotype-matched Ig with irrelevant specificity, was obtained
from the hybridoma cell line GL113 (DNAX). The IFN--specific IgG1
mAbs were obtained from the hybridomas AN18 (DNAX) and R4-6A2 (American
Type Culture Collection, Manassas, VA). Hybridomas were grown in
ascites in Harlan Sprague Dawley nude mice primed with IFA.
Nonimmune rat IgG were obtained from serum of Lewis rats. Abs were
purified by affinity chromatography using a protein G-agarose column,
followed by dialysis against PBS. FITC-conjugated rat anti-mouse
CD4, PE-conjugated rat anti-mouse CD8, and PE-conjugated rat
anti-mouse CD3 were purchased from PharMingen (San Diego,
CA).
Animals and infections
Specific pathogen-free female BALB/c mice were purchased from Harlan Iberica (Barcelona, Spain). Lewis rats and outbred Harlan Sprague Dawley nude mice were purchased from Gulbenkian Institute for Science (Oeiras, Portugal). 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.
M. avium 2447 SmT (an AIDS isolate obtained from F. Portaels, Institute of Tropical Medicine, Antwerp, Belgium) stored as frozen aliquots at -70°C as well as the inoculum used in experimental infections were prepared as described elsewhere (14). BALB/c mice were infected i.v. with 106 CFU of M. avium strain 2447 SmT through a lateral tail vein. At different time points, mice were sacrificed by cervical dislocation, and the organs (spleen, liver, and lung) 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 1–2 wk at 37°C, and the number of CFU was counted. The data are expressed as the mean of the log10 number of CFU recovered per organ ± 1 SD (n = 4 or 5 animals). Statistical significance of the differences was analyzed by performing the Student’s t test using unpaired data.
Treatment of mice with mAbs
To block the IL-10R, mice were injected i.p. with 1 mg
anti-IL-10R mAb 12 h before infection or 30 days after
infection, followed by i.p. injections with 0.2 mg anti-IL-10R mAb
every other day during the whole course of infection. Control mice were
submitted to the same protocol of administration of Abs, being injected
with anti--galactosidase mAb (GL113 mAb) as an Ig isotype
control or with nonimmune rat IgG.
Antibiotic regimens
Drug therapy was done orally by adding clarithromycin (200 mg/kg/day), rifabutin (40 mg/kg/day), and ethambutol (25 mg/kg/day) in the drinking water. Antibiotic treatment was done in one-half of the animals at the beginning of infection, and the remaining mice received the same therapy regimen 30 days after infection. The treatment was done until the end of the experimental period of infection. Control mice drank nonsupplemented water. The treatment of mice with mAbs was done in the same period of time as the antibiotic chemotherapy.
Ag preparations
Preparation of M. avium envelope proteins was performed as described elsewhere (38). Briefly, M. avium 2447 was grown for 2 wk in Sauton medium supplemented with 0.5% pyruvate and 0.5% glucose. The pellet obtained by centrifugation was resuspended in PBS containing 0.1% Tween 80, 1 mM MgCl2 (Merck, Darmstadt, Germany), and 1 mM benzamidine (Sigma), and sonicated with pulses of 1 min at maximum power, keeping the sample in ice during the procedure. The sonicate was centrifuged to discard intact mycobacteria (30 min at 2700 x g), and the supernatant was dialyzed against PBS. The latter was then ultracentrifuged for 2 h at 150,000 x g, and the pellet, containing the envelope proteins, was resuspended in PBS.
Purification and culture of CD4+ T cells
Spleens from mice noninfected or that had been infected for 20
days with M. avium 2447 treated either with anti-IL-10R
mAb or nonimmune rat IgG were aseptically collected. Half of the organ
was used to prepare single cell suspensions. Spleen cells were depleted
of RBCs by incubation in hemolytic buffer (155 mM
NH4Cl, 10 mM KHCO3,
pH 7.2) for 5–10 min at room temperature and thoroughly washed.
Nucleated spleen cells were pooled (n = 4 animals) and
incubated (5 x 106 cells/ml) in tissue
culture petri dishes at 37°C in a 7% CO2
atmosphere for 3 h to remove adherent cells. Nonadherent cells
were centrifuged; resuspended in PBS containing 0.1% glucose, 1% BSA,
and 5 mM EDTA, pH 7.2, at a concentration of 1 x
108 cells/ml; and incubated with microbeads
coated with anti-CD4 mAbs (clone L3T4; Miltenyi Biotec, Bergisch
Gladbach, Germany) at 4°C for 20 min. Cells were washed and
resuspended in PBS-glucose-BSA-EDTA buffer (108
cells/500 µl), and CD4+ T spleen cells were
positively selected by using MiniMACS separation columns (Miltenyi), as
described in the instructions from the manufacturer.
CD4+ T cells (94–95.2% pure on FACS analysis)
were washed, counted, and resuspended in RPMI 1640 containing 10%
heat-inactivated FCS, HEPES (10 mM), penicillin (100 U/ml),
streptomycin (100 µg/ml), L-glutamine (1%),
and 2-ME (0.05 mM). CD4+ T cells were cultured in
triplicate at a density of 1 x 105
cells/well in duplicate 96-well plates with irradiated (3000 rad)
nucleated spleen cells as APC (1 x 107
cells/ml) either with no further stimulus or in the presence of Con A
(5 µg/ml) or M. avium envelope proteins (4 µg/ml) at
37°C in 7% CO2 atmosphere. Supernatants were
collected from triplicate cultures after 72 h and frozen at
-20°C until the measurement of IFN- by ELISA. The duplicate
plates were used in the proliferation assay.
Analysis of the state of activation of peritoneal exudate cells (PEC)
PEC were obtained by washing with cold PBS the peritoneal cavity of noninfected mice or of mice that had been infected for 20 days with M. avium 2447 and treated with either anti-IL-10R mAb or nonimmune rat IgG as a control. Cells were washed and resuspended in DMEM containing 10 mM HEPES buffer, 1 mM sodium pyruvate, 1% L-glutamine, and 10% of heat-inactivated FCS, and total cell counts were done. PEC were plated in triplicate at a density of 4–5 x 105 cells/well in 96-well flat-bottom plates. After 3 h of incubation (37°C, 7% CO2), the nonadherent cells were removed by washing three times with prewarmed DMEM. Adherent cells were stimulated for 72 h with LPS (2 µg/ml) at 37°C in a 7% CO2 atmosphere to study their ability to produce nitrite. The amount of nitrite secreted by the adherent cells and present in supernatants was measured with the Griess reagent, as described elsewhere (39). Briefly, 100 µl Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine, and 2.5% H3PO4 in distilled H2O) was added to 100 µl cell-free supernatant. After 10 min of incubation at room temperature, the OD550 was read with an ELISA reader, and the nitrite concentration was determined by the use of a standard curve of NaNO2 concentrations.
Cell monolayers were lysed by three cycles of freezing and thawing, and the amount of protein in each well was determined by the Micro BCA Protein Assay reagent kit (Pierce, Rockford, IL), according to the instructions of the manufacturer. The data are expressed as the mean of the amount nitrite present in supernatant per mg of adherent cell protein found in each well ± 1 SD of triplicates from four individual mice for each group.
Flow cytometry
To analyze the CD4+ T cell-enriched spleen cells from uninfected and infected mice used in culture experiments, 106 cells from the purified CD4+ T cell pools were incubated for 30 min at 4°C in a microtiter plate with PE-conjugated rat anti-mouse CD8 or PE-conjugated rat anti-mouse CD3 and FITC-conjugated rat anti-mouse CD4 mAbs. Cells were washed twice with staining medium (PBS containing 0.1% sodium azide and 3% FCS) and resuspended in staining medium containing propidium iodide to allow exclusion of dead cells. Flow cytometric analysis was performed with a FACScan apparatus (Becton Dickinson, Mountain View, CA) equipped with PCLysisII software. For each sample, 10,000 events were analyzed.
Cytokine assays
Blood from sacrificed mice was allowed to clot, and serum was
collected after centrifugation and frozen at -70°C until use. The
spleen and lung homogenates were mixed with 0.1% of Triton X-100 (1:1)
and incubated for 2 h at 4°C. After centrifugation, supernatants
were collected and frozen at -70°C until use. Quantification of
IFN- in sera, culture supernatants, and homogenates of spleens and
lungs was done by a two-site sandwich ELISA with the
anti-IFN--specific affinity-purified mAbs (R4-6A2 as capture and
biotinylated AN-18 as detecting Abs). The sensitivity of the assay was
80 pg/ml.
Proliferation assay
Cell proliferation was measured by [3H]TdR incorporation. Briefly, proliferative responses were assessed after 48 h of culture in a humidified atmosphere of 7% CO2 in air. Cultures were pulsed 18–20 h before harvesting with 0.5 µCi [3H]TdR (Amersham, Little Chalfont, U.K.) per well, and incorporation of [3H]TdR was measured by liquid scintillation. Results were calculated as mean cpm of triplicate cultures ± 1 SD (n = 4 animals).
Immunization studies
Culture filtrate proteins of M. avium 2447 (CFP) were obtained from supernatants of M. avium 2447 cultures, as described elsewhere (38). To immunize animals, groups of eight mice were injected s.c. with 45 µg CFP three times at the base of the tail in separate locations, at weekly intervals. CFP was administered either as a solution or emulsified with 250 µg dimethyl dioctadecyl ammonium chloride (DDA; Eastman Kodak, Rochester, NY) as an adjuvant. The control mice were treated with DDA or PBS. Half of the animals in each group received 1 mg anti-IL-10R mAb i.p. 2 h before each immunization, whereas the other half was treated with nonimmune rat IgG as a control. Two months after the last immunization, mice were challenged with 106 CFU of M. avium 2447. The resistance provided by the experimental vaccination was calculated by quantifying the bacterial load in the spleen and the liver homogenates 30 days later, as described above. Values are reported as log10 protection calculated by subtracting the mean of log10 CFU in the immunized mice from the mean of log10 CFU in untreated mice. Statistical significance of the differences was analyzed by performing ANOVA test.
Statistical analysis
Statistical analysis was performed using Student’s t test or the ANOVA test (*, p < 0.05; **, p < 0.01).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the effect of blocking the IL-10R during M.
avium infections, BALB/c mice were infected with M.
avium 2447 and treated throughout the infection with either
anti-IL-10R mAb or nonimmune rat IgG, as described in
Materials and Methods. The growth of M. avium was
monitored for 90 days in the spleen, liver, and lung of infected
animals. As shown in Fig. 1
A,
the treatment with the blocking Ab had a small impact on the course of
the infection, leading to an increased resistance 1 mo after infection
in the liver (0.79 log10 less mycobacteria) and
spleen (0.55 log10 less mycobacteria), which
disappeared later in the chronic phase of the infection.
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is essential in the resistance against infection by
M. avium 2447 (40, 41) and that IL-10 has been
described as an antiinflammatory cytokine with an ability to indirectly
inhibit IFN- production (17, 18, 19), we evaluated the
levels of the latter cytokine in the sera and in homogenates of spleens
and lungs from infected mice. As observed in Fig. 1
B,
treatment with the anti-IL-10R mAb increased the levels of IFN-
at different time points of infection as compared with control mice.
This was particularly evident in serum at day 30 of infection and in
spleen at day 60 of infection, in which differences in the amount of
IFN- between both groups were more than 2-fold.
To analyze the importance of IL-10 in the late phase of infection, mice
were infected with M. avium and treated with either
anti-IL-10R mAb or control IgG starting on day 30 of infection. In
this experiment, no differences in the bacterial loads were found
between the two groups of animals at 90 days of infection either in
liver, spleen, or lung (Fig. 1C).
Effect of anti-IL-10R mAb treatment on the immune response
The previous data suggested that the major effect of
anti-IL-10R mAb treatment occurs early, i.e., in the first month of
infection. Thus, we infected mice with M. avium and treated
them with either anti-IL-10R mAb or nonimmune rat IgG as control
for 20 days. The animals were then sacrificed, and
CD4+ T cell function as well as macrophage
activation were studied. As shown in Fig. 2A, CD4+
T cells from spleens of mice treated with anti-IL-10R mAb produced
significantly increased amounts of IFN- in response to specific
mycobacterial proteins when compared with cells from control-infected
animals. Proliferative responses to M. avium proteins were
identical for CD4+ T cells from spleens of both
groups of infected mice (data not shown). FACS analysis showed that the
number of CD4+ T cells in culture was similar for
all groups of mice (9.5 x 104 cells for
noninfected mice; 9.4 x 104 cells for
anti-IL-10R mAb-treated infected mice; and 9.4 x
104 cells for control-infected mice). These data
suggest that the increment in the resistance to M. avium
infection as a result of blocking the IL-10R in the early phase of the
infection could be at least partially due to the increased priming of
CD4+ T cells for IFN- secretion.
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B, treatment of infected mice with
anti-IL-10R mAb also increased the activation of macrophages
explanted from the peritoneal cavity of infected mice. Peritoneal
macrophages from infected mice treated with anti-IL-10R mAb
produced 40-fold higher amounts of nitrite than those from infected
control animals. These data may indicate that treatment with
anti-IL-10R mAb in the early phase of infection augmented
macrophage functions other than the production of reactive nitrogen
intermediates, namely those involved in the mechanisms of resistance to
M. avium infection. Anti-IL-10R mAbs improve the efficacy of chemotherapy in refractory MAC infections
Given the effects of anti-IL-10R treatment on macrophage
activation, we analyzed whether this treatment had a synergistic effect
on antimicrobial therapy. BALB/c mice were infected with M.
avium, and half of them were given a combination of antibiotics in
one of two regimens. In the first, chemotherapy was started at the
beginning of infection (Fig. 3A), whereas in a second
protocol, chemotherapy started only after 1 mo of infection (Fig. 3B). The antibiotics were given in combination either with
control IgG1 from hybridoma GL113 or with anti-IL-10R mAb. As shown
in Fig. 3A, chemotherapy alone was effective when started
early in infection. In that situation, anti-IL-10R mAb therapy did
not improve the effectiveness of chemotherapy (Fig. 3A).
When antibiotic therapy was started 1 mo after the inoculation of the
mycobacteria, it was without any effect in the liver and had a minor
effect in the spleen and lung (Fig. 3B). As previously
observed, treatment of mice with anti-IL-10R alone starting at 1 mo
of infection had also no effect in the liver and lung and caused a very
small decrease in the spleen. However, the treatment with
anti-IL-10R Abs reverted the refractoriness to chemotherapy of
these chronically infected animals. Indeed, mice under this combined
therapy showed a significant reduction in bacterial loads in the lung
(1.21 log10), in the spleen (0.97
log10), and in the liver (0.56
log10) as compared with mice treated with GL113
mAb without chemotherapy. Additionally, the combined therapy was
effective in the clearance of bacteria in both liver and spleen when
compared with bacterial loads found in mice at 30 days of infection,
the starting point for the treatment (liver p = 0.000
and spleen p = 0.003).
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Since we found that anti-IL-10R treatment would enhance the
priming of presumably protective, IFN--secreting
CD4+ T cells, we evaluated the usefulness of the
anti-IL-10R mAb administration on the design of antimycobacterial
vaccines. We used secreted proteins from M. avium as the Ag
source. This choice was based on previous studies from our lab that
showed the potential of CFP in the induction of a protective immunity
to M. avium (14). Furthermore, the protection
against M. avium infection conferred by the subunit vaccine
composed of CFP emulsified in an adjuvant had the same magnitude as
that conferred by BCG (15). Mice were immunized with CFPs
either alone or admixed with DDA as an adjuvant. As controls for the
nonspecific inflammatory-dependent induction of resistance to
infection, mice were injected with DDA or PBS. To test whether blocking
the IL-10R during the immunization had an effect on the resistance
provided by the subunit vaccines, mice were injected with
anti-IL-10R mAb or with rat nonimmune IgG as control 2 h
before every dose of the subunit vaccines. As shown in Fig. 4, the combination of CFP and DDA induced
significant protection in mice against a challenge with the homologous
M. avium strain. CFP alone, on the other hand, failed to
protect mice. However, treatment of mice with anti-IL-10R mAb
during vaccination made CFP alone immunogenic, leading to the induction
of protective immunity. The treatment with the anti-IL-10R mAb did
not improve the efficacy of the vaccine consisting of CFP plus DDA
(Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 3). These data contrast with
previously published experiments in which anti-IL-10 Abs were used
and which described major effects of the treatment on the course of
infection (24, 25). These differences may be related to
the different strains of M. avium used (24, 25), the differences in the route of infection
(24), or the strains of mice studied (24). In
our hands, the anti-IL-10R Abs had little impact on the outcome of
infection, but still led to significant differences in mycobacterial
loads. Previous work from our laboratory, using anti-IL-10 mAbs,
failed to show any effects on the course of infection
(43). Also, we have recently found that IL-10
gene-deficient mice were only marginally more resistant to infection by
the strain of M. avium used in this study than control mice
(44), mimicking the data reported in this work. Overall,
we are confident to state that IL-10 plays a minor role in determining
susceptibility to infection by M. avium, although some
protection may be found at early stages of infection by blocking its
activity. These data are similar to those obtained with experimental
therapies with rIL-12 in which the immunomodulatory interventions
accelerated the immune response by increasing the levels of IFN- in
early phases of infection (14), but did not dramatically
affect the overall outcome of the infection (14, 45).
IL-10 has been described in the literature as a potent
macrophage-deactivating factor by suppressing activation of macrophages
and dendritic cells, inhibiting their ability to both secrete cytokines
and express costimulatory molecules at their surface
(17, 18, 19). In our study, we found that the minor protection
afforded by treatment with anti-IL-10R mAb observed in the early
phase of M. avium infection was associated with an increase
in both the priming of CD4+ T cells to produce
IFN- and activation of some functions of the macrophages (Fig. 2).
Indeed, IFN- is important in an early phase of the infection for the
induction of protective immune mechanisms that lead to
mycobacteriostasis (14, 40, 41). Later, the increment in
IFN- production induced by anti-IL-10R mAb treatment did not
improve the resistance to M. avium, in agreement with our
previous work in which neutralization of IFN- after 30 days of
infection had a minor effect on bacterial loads (41).
Thus, the effects of the treatment with anti-IL-10R mAb on the
course of a primary infection may relate to a combination of the
effects on the emergence of protective T cells producing IFN- and
the promotion of the macrophage activation.
Our data highlight the potential of an anti-IL-10R treatment to revert the refractoriness of the infections by MAC to chemotherapy. We observed that once the infection by M. avium was established, a combination of antibiotics, which was effective when administered early in infection, became unable to clear the infection. This is a situation that corresponds more closely to the clinical situation in which patients are diagnosed when already heavily infected by nontuberculous mycobacteria (3). However, when antibiotic chemotherapy was associated with anti-IL-10R treatments, the effectiveness of the therapy was regained in those chronic infections. This contrasted with the lack of effects of the mAb therapy when chemotherapy was started at the time of infection. Thus, the combination of therapeutic strategies aimed at interfering with IL-10 signaling with classical chemotherapy may prove of importance in the management of human infections by MAC. In this regard, published data suggest that increased IL-10 production may contribute to a predisposition to M. avium infections in AIDS patients (46, 47, 48). It would also be of interest to study such an approach in the therapy of tuberculosis, as it was shown that immune cells from tuberculosis patients produce IL-10 and that anergy in tuberculosis patients was related to T cell-derived IL-10 (49, 50, 51). Curiously, similar to our present findings, IL-10 seems not to underlie the susceptibility to tuberculosis in the mouse model (52). Since long-term treatment of both tuberculosis and M. avium infection with the appropriate conventional chemotherapy usually leads to severe side effects (11, 12), the possibility of improving antimycobacterial activity of current antibiotics by interfering with IL-10 signaling may help circumvent these problems.
The mechanisms underlying the effects of the anti-IL-10R treatment
on chemotherapy are still unknown, but it is likely that IL-10 being
produced at the peak of the immune response blocks the activity of the
antibiotics by deactivating the macrophage. Indeed, the effects of
anti-IL-10R treatment on macrophage functions are more pronounced
than those on T cell activity. How IL-10 interferes in the
intramacrophagic action of antibiotics is currently being studied. On
the other hand, the data obtained in this study with anti-IL-10R
mAbs are reminiscent of the data obtained by Doherty and Sher
(45), with the association of chemotherapy with rIL-12
therapy. In that work, a synergistic activity was found between the
cytokine and the antimicrobial compounds, which was dependent on
IFN- production. On the other hand, clinical trials have found that
IFN- may improve the antibiotic chemotherapy of MAC infections
(53). Thus, it is possible that the synergistic activity
of anti-IL-10R mAb and antibiotic therapy may depend on both an
increased IFN- production and an enhanced response by the macrophage
to this cytokine in a cytokine cascade most likely also involving
increased IL-12 production.
The interference with IL-10 may also prove of interest in the design of new vaccines, namely those using subunit components of pathogens. Our data showed that anti-IL-10R mAb could substitute for the adjuvant in the induction of protective immunity. However, it should be stressed that much more research in this area should be done to ascertain the safety of such strategies. The mechanism of action is unclear, but it may be related to the one described by Castro et al. (36). In their system, LPS was required in order for soluble protein Ag to induce a Th1 response when IL-10 activity was abrogated. Our Ag preparations include not only proteins, but probably other mycobacterial components present in trace amounts, such as glycolipids, that could be recognized by macrophages and/or dendritic cells through the pattern-recognition receptors, eliciting both a proinflammatory immune response (54, 55, 56, 57) and dendritic cell maturation (58). Thus, neutralization of IL-10 may augment the adjuvant effect of these contaminants in the subunit vaccine. The adjuvant effects of anti-IL-10R mAb may relate to the increased expression of costimulatory molecules and class II MHC (17, 18, 19) as well as to effects on APC, such as dendritic cells, since the biology of the latter is strongly influenced by IL-10 (58, 59, 60).
In conclusion, although treatments with anti-IL-10R mAb alone may not improve the outcome of mycobacterial infections, they may be useful in combination with antibiotic chemotherapy as well as in the field of vaccination as adjuvants in subunit vaccines.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rui Appelberg, Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-180 Porto, Portugal. E-mail address: rappelb{at}ibmc.up.pt
3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; CFP, culture filtrate proteins of M. avium 2447; DDA, dimethyl dioctadecyl ammonium chloride; MAC, M. avium complex; PEC, peritoneal exudate cell.
Received for publication February 26, 2001. Accepted for publication May 17, 2001.
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. N. Engl. J. Med. 330:1348.This article has been cited by other articles:
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S. Roque, C. Nobrega, R. Appelberg, and M. Correia-Neves IL-10 Underlies Distinct Susceptibility of BALB/c and C57BL/6 Mice to Mycobacterium avium Infection and Influences Efficacy of Antibiotic Therapy J. Immunol., June 15, 2007; 178(12): 8028 - 8035. [Abstract] [Full Text] [PDF]
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 D. K. Flaherty, B. Vesosky, G. L. Beamer, P. Stromberg, and J. Turner Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination J. Leukoc. Biol., December 1, 2006; 80(6): 1262 - 1271. [Abstract] [Full Text] [PDF]
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M. H. Shaw, G. J. Freeman, M. F. Scott, B. A. Fox, D. J. Bzik, Y. Belkaid, and G. S. Yap Tyk2 Negatively Regulates Adaptive Th1 Immunity by Mediating IL-10 Signaling and Promoting IFN-{gamma}-Dependent IL-10 Reactivation. J. Immunol., June 15, 2006; 176(12): 7263 - 7271. [Abstract] [Full Text] [PDF]
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 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]
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J. Turner, M. Gonzalez-Juarrero, D. L. Ellis, R. J. Basaraba, A. Kipnis, I. M. Orme, and A. M. Cooper In Vivo IL-10 Production Reactivates Chronic Pulmonary Tuberculosis in C57BL/6 Mice J. Immunol., December 1, 2002; 169(11): 6343 - 6351. [Abstract] [Full Text] [PDF]
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) or nonimmune rat IgG as
a control (
and 
). Treatment of mice was started at the beginning
(A and B) or at day 30 (C)
of infection, and was done until the end of experimental M.
avium infection. A and C,
Enumeration of viable bacteria recovered from the organs of infected
mice at different time points of infection. Each time point represents
the geometric mean of the CFU from four or five animals. The
statistical significance of the decrease in bacterial load in
anti-IL-10R mAb-treated mice compared with control mice is labeled
with asterisks (**, p < 0.01).
B, IFN-
) or
infected mice treated (
