| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Nevertheless, IFN- itself, even
when given at a higher dose than IL-12, failed to significantly enhance
antibiotic clearance of bacteria. Together these findings suggest that
IL-12 may be a particularly potent adjunct for chemotherapy of M.
avium infection in immunocompromised individuals and may result
in more effective control of the pathogen without the need for
increased drug dosage. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As with most intracellular infections, the control of M.
avium in immunocompetent hosts appears to depend upon the
induction of cell-mediated mechanisms as opposed to humoral immune
mechanisms (16, 17, 18). Thus, in both human disease (19, 20) and murine
experimental models, M. avium infection stimulates the
production of macrophage-activating lymphokines (IFN-, TNF-
) as
well as microbicidal products such as nitric oxide and/or reactive
oxygen intermediates (21, 22, 23, 24, 25, 26). That such responses are important in the
control of infection is evidenced by the increased bacterial growth
seen in mice that are deficient in T cells or the production of IFN-
(24, 27, 28, 29) or in patients with defects in the generation or effector
functions of Th1 responses (30). Studies in mice treated with
neutralizing mAb to IL-12 also show decreased resistance to the
pathogen (22, 31). The latter cytokine is thought to play a pivotal
role in the induction of most IFN--dependent, cell-mediated
responses (32). It is presumed that the loss of control of M.
avium infection in late-stage AIDS patients reflects an impairment
in those CD4-dependent effector mechanisms that are similar to those
studied in vitro and in experimental laboratory hosts (5).
In a previous study, we systematically compared the growth of M.
avium in mice with genetically engineered defects in lymphokine
synthesis and macrophage effector function (24). Interestingly, T
cell-deficient SCID mice, while more susceptible to infection than
wild-type (wt)2 animals, were
nevertheless more resistant than comparably infected IFN--deficient
mice. This finding suggested that in the absence of T cells, M.
avium infection can be partially controlled by IFN- that is
derived from NK cells, which are known to serve as an alternative
source of this cytokine. Since IL-12 is an important stimulator of
NK-produced IFN- (33, 34), we have considered possibly using the
former cytokine to enhance resistance to the bacterium in T
cell-deficient hosts.
In the present report, we demonstrate that rIL-12 does indeed augment resistance to M. avium infection in SCID mice and, more importantly, acts synergistically to promote bacterial clearance when combined with conventional antibiotics. Our findings suggest that combined IL-12/drug treatment may offer a highly effective strategy for the management of atypical mycobacterioses in immunocompromised individuals.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 mice were obtained from Charles River Laboratories
(Wilmington, MA). C57BL/6-SCID/SzJ (BL/SCID) mice were initially
purchased from The Jackson Laboratory (Bar Harbor, ME). Mice that were
deficient for expression of the IFN- gene and backcrossed onto the
C57BL/6 background to the seventh generation were derived from stocks
originally provided by D. Dalton and S. Stewart (Genentech, San Bruno,
CA). These IFN- knockout (GKO) mice were genotyped by the PCR of
blood as previously described (35). Mice with targeted disruptions of
the IL-12 p40 gene (IL-12 knockout (KO)) were generously
provided by J. Magram (Hoffman-La Roche, Nutley, NJ). All animals were
maintained in specific pathogen-free conditions and housed in the same
animal facility at the National Institute of Allergy and Infectious
Diseases (NIAID); all mice were age- and sex-matched for experiments
and used at 6 to 10 wk of age.
Bacterial strain and Ag
The M. avium strain 2–151 SmT was kindly provided by Dr. A. Cooper (Colorado State University, Colorado). Stocks were prepared according to the following protocol for use in all subsequent experiments. BL/SCID mice were infected i.v. with 107 CFU, spleens were taken after 6 wk, and single-cell suspensions were prepared and plated on Middlebrook 7H11 agar (National Institutes of Health media unit, Bethesda, MD). Bacterial colonies were harvested from agar plates after 2 wk, dispersed by 2 x 5 s uses of a probe sonicator (Heat Systems, Farmingdale, NY), frozen in saline at 108 to 109 CFU/ml (determined after sonication), and stored frozen at -70°C.
Mycobacterial Ag (MAg) was made by freeze-thaw lysis of M. avium at 108 CFU/ml. Bacteria were quick-frozen in 0.5 ml aliquots in PBS (Life Technologies, Gaithersburg, MD) using an ethanol/dry ice mixture and subsequently thawed at 37°C. After three cycles, the lysate was disrupted by 3 x 10 s uses of a probe sonicator. The protein concentration was determined using the bicinchoninic acid assay (36) and adjusted to 2.5 mg/ml. MAg was used to stimulate spleen cells at a final concentration of 50 µg/ml.
Drugs and cytokine treatment
Mice were injected i.v. with 107 CFU of the
M. avium strain 2–151 SmT. After 2 wk, the animals had
developed a disseminated infection, and bacteria were readily
demonstrable in a variety of organs. Treatment was subsequently
initiated with either clarithromycin, rifabutin, IL-12, IFN-, or
combinations of drug and cytokine that were administered i.p. in 200
µl of saline at the doses indicated. Since neither rifabutin nor
clarithromycin is readily soluble in an aqueous solution, the former
was dissolved in DMSO, while the latter was wet with 95% ethanol. Both
were subsequently diluted to 1% in saline as suspensions in accordance
with the manufacturer’s instructions. Injections were repeated on
alternate days for three sequential doses. After a rest period of 2
days, another three doses were administered on the same schedule.
Control animals were given saline alone at the same time intervals.
Preliminary experiments, which tested drugs in the range of 5 to 200
mg/kg by several different routes (s.c., peritoneal, or gavage) on
daily or alternate day administration, indicated that this regimen was
highly efficacious while producing a minimum of apparent side effects
(data not shown). Mice were sacrificed for the analysis of infection
and immune responses at 2 days after the final injection.
In some experiments, mice were simultaneously treated with the
neutralizing anti-IFN- mAb (XMG.6) or with a control mAb (GL113)
(cell lines provided by R. Coffman, DNAX, Palo Alto, CA); the mAbs were
given i.p. at 1 mg/animal at the same time as the cytokine and drug
injections.
Measurement of bacterial infection in vivo
The progress of bacterial growth was monitored at various time
points by assaying bacterial CFUs in the spleen and lungs of infected
mice. Organs were weighed, and then a small portion of each tissue was
removed, embedded in paraffin for sectioning, and stained with
hematoxylin and eosin or the Fite acid-fast method (American Histolabs,
Gaithersburg, MD). The remainder of each organ was homogenized by
maceration through a fine mesh stainless steel sieve into complete RPMI
1640 (Life Technologies, Grand Island, NY) (including 2 mM glutamine,
30 µg/ml of ampicillin, 10% FCS, and 50 µM 2-ME); the cells were
counted, and an aliquot was diluted to 106/ml in
buffered saline for the enumeration of viable bacterial counts.
Triplicate, serial 10-fold dilutions of the cells were then performed
on Middlebrook 7H11 agar plates which were incubated at 37°C for 2 to
3 wk. Colonies were then counted, and the number of CFUs per organ were
calculated. The detection limit of this protocol is 
100 CFU/sample,
although the precise limit is defined by the number of cells contained
in the target organ, as this number is typically better in early
infection (as low as 10 CFU/sample).
Measurement of IFN- expression
The relative level of IFN- mRNA in spleen cells was assayed
by RT-PCR as previously described (37). Briefly, single-cell
suspensions that had been prepared as described above were thoroughly
washed, and total RNA was prepared by lysis of 106
cells in RNA-STAT-60 (Tel-Test ‘B’, Friendswood, TX) followed by
precipitation from the aqueous phase as recommended by the
manufacturer. Recovered RNA was resuspended in diethyl
pyrocarbonate-treated, distilled, deionized water and cDNA synthesized.
PCRs were performed on serial dilutions of cDNA (from 10 µl) in a
final volume of 50 µl, and a sample (10 µl) of each PCR reaction
was electrophoresed through a 1.0% agarose gel and visualized with
ethidium bromide. The number of cycles of PCR amplification used was
first determined by amplifying cDNA through 24 to 36 cycles and
comparing the product obtained to a standard curve from LPS-stimulated
spleen cells. The number of cycles of amplification was chosen to give
a PCR product that was easily detected in a gel while remaining on the
linear part of the amplification curve. Ethidium bromide-stained gels
were photographed with an Eagle Eye II Still Video System (Stratagene,
La Jolla, CA), and the intensity of fluorescence was determined using
the associated Eaglesight software. To ensure that equivalent amounts
of cDNA were used in each reaction, PCR was performed for hypoxanthine
phosphoribosyltransferase (HPRT) from each sample, and the cDNA was
adjusted to equivalent levels. Each pair of primers spanned at least
one intron, which allowed mRNA to be distinguished from any
contaminating genomic DNA, and all primers were synthesized at NIAID.
The cycle number and sequences used were: HPRT (30 cycles): HPRT sense
GTT GGA TAC AGG CCA GAC TTT GTT G, HPRT antisense GAG GGT AGG CTG GCC
TAT AGG CT; IFN- (29–30 cycles): IFN- sense TGG AGG AAC TGG CAA
AAG GAT GGT, IFN- antisense TTG GGA CAA TCT CTT CCC CAC.
IFN- protein levels were also assayed after the restimulation of
spleen cells in vitro. Briefly, splenocyte suspensions (2 x
106/ml) in complete RPMI 1640 medium were distributed in
96-well plates (Costar, Cambridge, MA) and exposed to MAg at 50 µg/ml
in a total culture volume of 200 µl. Supernatants were collected at
72 h, and IFN- levels were assayed by a two-site sandwich ELISA
as previously described (38).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In a previous study of genetically immunodeficient animals, we
found that both GKO and BL/SCID mice are defective in their control of
chronic M. avium infection (24). To comparatively assess the
contribution of IL-12 to host resistance, we studied the growth of the
organism in mice lacking a functional IL-12 p40 gene (39, 40). These animals were each i.v. inoculated with 107 CFU
of the virulent 2–151 SmT strain. As positive or negative controls, we
simultaneously infected BL/SCID, GKO, or wt mice that also share the
same C57BL/6 genetic background. Animals were sacrificed at regular
intervals after inoculation, and the growth of the bacteria in spleens
was determined by the titration of single-cell suspensions onto 7H11
agar and expressed as bacterial load per whole organ. Qualitatively
similar results were obtained when the data were expressed as bacterial
load either per gram of tissue or per million cells (data not shown).
In addition, RT-PCR was performed on RNA that had been extracted from
spleen cells to measure the expression of the IFN- gene.
As expected on the basis of previous studies, all of the mice strains
tested showed an initial increase in bacterial numbers after infection
(Fig. 1
, top panel). In
C57BL/6 mice, growth slowed as the infection was gradually brought
under control. As described previously, this correlated with both the
induction of a strong IFN- response and the development of
significant pathology, which were characterized by a monocytic
infiltration of infected organs and an enlargement of the liver and
spleen (24, 41). Bacterial numbers in the spleens of IL-12 KO, GKO, and
BL/SCID mice, in contrast to the C57BL/6 control mice, continued to
increase up to 8 wk postinfection (Fig. 1, top
panel). Infected IL-12 KO and GKO mice displayed nearly
identical growth kinetics, confirming the critical function of both
cytokines in the development of resistance. Consistent with the known
role of IL-12 in the induction of IFN- (42), spleen cells from the
infected IL-12 KO mice produced IFN- mRNA in much lower quantities
than did the equivalent cell populations from the infected control
animals (Fig. 1, bottom panel). Interestingly,
infected BL/SCID mice showed bacterial growth kinetics that were
distinct from both IL-12 KO and GKO animals, displaying an intermediate
pattern of susceptibility (Fig. 1, top panel). A
similar difference in infection was seen in the lungs of these strains,
where again, BL/SCID mice characteristically developed bacterial loads
that were intermediate between the wt C57BL/6 and the gene KO mice
(data not shown). Furthermore, spleen cells from infected BL/SCID mice
produced IFN- mRNA at levels that were intermediate between that
synthesized by splenocytes from infected wt and IL-12 KO animals. FACS
analysis and in vitro restimulation with Ag failed to show any evidence
for the development of a specific T cell response in BL/SCID mice (data
not shown), indicating that the partial resistance of these animals
cannot be accounted for by "leakiness" of the SCID mutation.
Instead, the above observations suggested that the residual control of
bacterial growth in BL/SCID mice is due to the production of IFN-
from a non-T cell (i.e., NK cell) source, a response which is
characteristically dependent upon IL-12 (43).
|
To directly assess whether IL-12 can mediate resistance in T-deficient hosts, BL/SCID mice were infected i.v. with 107 virulent M. avium and 2 wk later treated for an additional 2 wk with 500 ng/mouse of exogenous murine rIL-12 that was delivered i.p. in 200 µl of buffered saline on a daily basis. This protocol resulted in a significant (p < 0.05) reduction in splenic bacterial colony counts vs those measured in control infected animals that had been injected with saline alone (mean CFU ± SE from two experiments: IL-12 group = 1.3 ± 0.27 x 104; control group = 6.8 ± 0.64 x 104). Although they survived the treatment, the mice receiving IL-12 at this dose displayed noticeable wasting and splenomegaly when compared with control animals (data not shown).
Although the effect of the high-dose IL-12 treatment on M. avium infection in BL/SCID mice was not dramatic, we hypothesized that the cytokine might induce a more marked increase in bacterial clearance when combined with known antimycobacterial drugs. In performing this analysis, the frequency of IL-12 administration and consequently the total dose was reduced by half (i.e., to 500 ng/mouse on alternate days) to minimize side effects and simplify the treatment regimen. When administered at this dose, IL-12 caused no mortality, weight loss, or change in outward appearance of the animals in the study, while still resulting in a noticeable increase in spleen weight (data not shown). Clarithromycin, a macrolide antibiotic that has been shown to be effective against M. avium infections in both human and mouse (44, 45) was used in the experiment and administered i.p. simultaneously with the IL-12. The dose of the drug chosen (200 mg/kg) was considered to be optimal according to preliminary experiments.
As shown in Figure 2, top
panel, treatment with IL-12 at the lower dose caused only a minor
reduction in splenic bacterial counts vs the recoveries from control
mice. In contrast, when combined with clarithromycin, even the lowered
IL-12 treatment resulted in a dramatic decrease in splenic bacterial
colonies, >100-fold compared with the numbers determined in control
animals. This reduction was highly significant
(p < 0.001) with regard to the clearance of
bacteria induced by the drug alone at this dose. The same synergistic
effect of combined IL-12/clarithromycin treatment was reproducibly
observed in four separate experiments.
|
, top panel, combined
IL-12/rifabutin administration again resulted in a >100-fold decrease
in colony counts which was significant (p <
0.01) vs that induced by the drug alone.
|
, top
panel). In contrast, synergy with IL-12 was evident only at
the highest dose of rifabutin tested because of the lower efficiency of
this drug (Fig. 4, bottom panel). Together, these
findings indicated that IL-12 can enhance the efficacy of M.
avium chemotherapy in immunodeficient hosts, and that this effect
is not a peculiarity of the specific drug or regimen employed.
|
and the
bottom panel to Figure 3, combined IL-12/drug treatment was
also highly effective in promoting bacterial clearance in
immunocompetent C57BL/6 mice; in the case of animals receiving
clarithromycin, this treatment reduced bacterial counts to below the
level of detection (<1 CFU per 104 spleen cells). Thus,
the protocol can also be efficacious in hosts that are already mounting
an immune response to the infection.
Dependence of synergistic effect of IL-12 treatment on IFN-
To assess the role of IFN- induction in the combined effect of
IL-12 and drugs on M. avium infection in immunodeficient
hosts, we simultaneously administered neutralizing anti-IFN- mAb
to IL-12/clarithromycin treated, infected BL/SCID mice. As shown in
Figure 2, top panel, this procedure completely ablated the
contribution of IL-12 to the reduction in splenic colony counts that
were induced by combined cytokine/drug therapy, while animals receiving
control Abs were unaffected. Moreover, combined IL-12/clarithromycin
treatment failed to augment bacterial clearance beyond the level
induced by drug alone in IFN--deficient (GKO) mice (Fig. 2, bottom panel). Interestingly, treating GKO mice with
IL-12 in the absence of drug caused a significant enhancement of
bacterial recovery. This effect is most likely due to the noticeable
influx of macrophages into the spleens of IL-12-treated animals, which,
in the case of GKO mice, would allow expanded growth of the pathogen in
the absence of immune control.
To confirm that IFN- was indeed induced in mice treated with IL-12,
mRNA that was specific for the cytokine was quantitated in the spleen
cells of infected BL/SCID mice by semiquantitative RT-PCR. In addition,
secretion of IFN- by the same spleen cells was measured after
restimulation with MAg in vitro. As shown in Figure 5, levels of IFN- message and protein
were significantly augmented in animals treated with IL-12 alone or
with IL-12 plus clarithromycin. In both cases, this increase was
partially blocked by the in vivo injection of anti-IFN- mAb,
suggesting a role for the cytokine in maintaining levels of IL-12 and,
consequently, IFN- itself in infected hosts. When administered in
combination with IL-12, clarithromycin caused a moderate decrease in
IFN- message and protein levels compared with that seen with IL-12
treatment alone, perhaps due to reduced induction of the lymphokine
because of the drop in bacterial numbers.
|
for IL-12 results in a loss of therapeutic
efficacy
Since the effect of IL-12 on drug-induced bacterial clearance is
dependent upon IFN-, we also evaluated the therapeutic activity of
the latter cytokine in the same antibiotic treatment protocol. In these
experiments, a dose of IFN- (1.0 µg/mouse/injection) that was
twice that of IL-12 was employed. As shown in Figure 6, this higher dose of IFN- failed to
promote bacterial clearance in GKO mice, suggesting a requirement for
greater levels of the cytokine to reconstitute the animals.
Interestingly, IFN- also failed to show any beneficial effects in
C57BL/6 mice, perhaps since endogenous IFN- levels were already very
high in this strain (Fig. 1, bottom panel). In
contrast, IFN- alone did have a small but significant therapeutic
benefit in BL/SCID mice, possibly by augmenting the intermediate levels
of the cytokine expressed by that strain (Fig. 6 and bottom
panel to Fig. 1). Nevertheless, in all three mouse strains tested,
IFN- administration clearly failed to enhance the clearance of
M. avium that was induced by the antibiotic (clarithromycin)
(Fig. 6). Thus, even when employed at a higher dose than IL-12, IFN-
cannot effectively substitute for that cytokine in promoting drug
therapy of M. avium infection.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present report, we have extended the concept of combined
IL-12/drug therapy to the treatment of an important opportunistic
pathogen, M. avium, in a T cell-deficient setting which is
similar to that expected in late-stage AIDS patients who are the
primary victims of atypical mycobacterioses. In contrast to the
previous studies cited above, the effects of IL-12 and drug observed in
the SCID mouse model we employed cannot be due to immunoregulatory
changes in T lymphocyte function but instead must involve the induction
of alternative pathways of resistance revealed in an immunodeficient
milieu. Previous work employing neutralizing mAb had suggested a role
for endogenous IL-12 in host resistance to M. avium (22, 31)
based on elevated bacterial counts. In the current study, this function
of IL-12 was confirmed in our model of M. avium infection
using mice with a defective IL-12 p40 gene. These animals
showed a >1000-fold increase in splenic bacterial loads and
importantly were indistinguishable from IFN--deficient mice in their
high susceptibility to infection (Fig. 1, top panel).
Moreover, both IL-12- and IFN--deficient animals became more heavily
infected than SCID mice, suggesting that the latter animals retain a
partially effective IL-12/IFN--dependent resistance mechanism.
Indeed, we have previously shown that infected SCID mice are only
partially impaired in their mRNA responses for both cytokines (24), and
their reduced production of IFN- message relative to wt mice was
confirmed here (Fig. 1, bottom panel). Interestingly,
IL-12 KO mice also retain a partial IFN- response but at a level
that is significantly lower than that observed in the SCID animals
(Fig. 1, bottom panel). Thus, it seemed logical to
assume that the defective resistance of SCID mice reflects a deficiency
in IL-12/IFN- production that might be corrected by the exogenous
administration of one of the cytokines.
When administered to previously infected SCID mice, rIL-12 in high doses (500 ng/mouse, daily) did indeed cause a fivefold reduction in splenic bacterial loads as measured 2 wk later. The use of higher doses and longer treatment periods was impractical given the limited supply of cytokine, although a larger reduction in CFUs was observed in a single experiment in which cytokine was given at the same dose for 4 wk (data not shown). These results support earlier observations (23) showing that exogenous IL-12 treatment augments the resistance of BALB/c and DBA/2 mice to M. avium infection and extend these findings by demonstrating that comparable effects on bacterial growth can be induced in the absence of functional T cells. Nevertheless, it is doubtful that long-term treatment with IL-12 would be economically practical or sufficiently efficacious to justify the use of such a regimen in AIDS patients with atypical mycobacterioses. Moreover, a clinical trial of such a procedure would require the removal of patients from antibiotic therapy, an unethical practice. The latter considerations further support the rationale for combined IL-12/drug therapy.
The major finding of the current study is that the treatment of
M. avium-infected, T cell-deficient mice with rIL-12 and
clarithromycin results in a striking reduction in splenic bacterial
loads. This effect was observed using a suboptimal, lowered total dose
of cytokine (500 ng/mouse, three times per week) and was clearly
greater than that induced by the drug or cytokine alone (Fig. 2).
Moreover, the same drug/cytokine synergy was observed in
immunocompetent C57BL/6 mice as well as with a different
antimycobacterial agent, rifabutin (Figs. 3 and 4), suggesting that
IL-12 coadministration may have broad application as an adjunct for
chemotherapy of M. avium infections.
Although our experiments in GKO mice and with IFN- neutralization
indicate that IL-12 mediates this synergistic effect through the
induction of the former lymphokine, the mechanism by which IFN-
enhances drug activity is not yet clear. An obvious explanation is that
drug treatment lowers the resistance of the organism to microbicidal
killing by IFN--activated macrophages or, conversely, that
IFN--induced macrophage activation prevents the recovery of bacteria
that have been partially crippled by drug treatment. Alternatively, as
suggested by the results of previous studies using IFN- in
combination with other antimicrobials (50), the cytokine may enhance
the uptake of antibiotics into infected cells. Although the
participation of one or several of the above mechanisms would seem
likely, other data suggest that synergy in the activity of IL-12 and
antibiotics in the therapy of M. avium may have a quite
different explanation. Thus, as reported previously (24), we have found
that the virulent bacterial strain used in our in vivo mouse infections
appears to be refractory to in vitro killing by IFN--activated
macrophages. Moreover, our preliminary attempts to demonstrate an
enhancement of drug (clarithromycin)-mediated intracellular killing of
M. avium in vitro by the addition of IFN- to the cultures
have failed to reveal significant effects (data not shown). Therefore,
it is possible that IL-12 augments bacterial clearance by an
IFN--dependent mechanism that is unrelated to the direct
microbicidal function of the macrophage.
The data demonstrating that IL-12 mediates its effects on M.
avium through the induction of IFN- raise the question of
whether IFN- itself would be equal or superior to IL-12 in
synergizing with antibiotics for bacterial clearance. To address this
issue, infected mice were treated with clarithromycin and IFN- at a
level that was twice that of the IL-12 employed in identically
performed drug/cytokine experiments. Interestingly, no significant
effects of IFN- on bacterial clearance were observed in the presence
of drug with that dose and regimen (Fig. 6), suggesting that IL-12 is a
more effective immunotherapeutic agent. Since it is known that IL-12
has a longer half-life than IFN- in vivo (51) and, as a upstream
inducer, should trigger larger amounts of the latter cytokine than the
dose injected, one possibility is that IL-12 administration results in
a higher, sustained level of IFN- than that achieved by direct
IFN- treatment. Alternatively, IL-12 is known to have several
physiologic effects (e.g., on hemopoiesis (41, 52) and nitric oxide
induction via TNF- (53)) that are not shared by IFN-, and it is
conceivable that one of these activities may synergize with the
IFN--mediated functions of the cytokine in promoting bacterial
clearance. Detailed dose-response studies comparing the effects of
IFN- and IL-12 administration on the host response to M.
avium in drug-treated animals will be required to address these
questions.
Although the mechanisms underlying the efficacy of combined IL-12/antibiotic treatment remain to be elucidated, our current findings suggest that the strategy is worthy of further investigation as an approach for improving the management of this important opportunistic pathogen in AIDS patients. While considerable toxicity was encountered in the initial clinical trials with IL-12, this problem can now be circumvented by administering the cytokine with a properly scheduled regimen (54). Therefore, IL-12-based combination therapy should be feasible in a clinical setting and could potentially lead to a treatment requiring lower doses of the poorly tolerated drugs that are presently used in M. avium chemotherapy. Finally, the same approach may be generally applicable to the management of other opportunistic infections (e.g., Toxoplasma, Histoplasma, and Candida) that are known to be sensitive to the effects of IL-12 in experimental models (55, 56, 57).
|
Acknowledgments |
|---|
, and the Genetics
Institute for supplying rIL-12. We are also grateful to Abbott
Laboratories and Pharmacia-Upjohn for donating the clarithromycin and
rifabutin employed in this study. Finally, we express our appreciation
to Drs. Steve Holland, Shukal Bala, and Henry Masur for their helpful
discussions and advice on mycobacterial therapy. |
Footnotes |
|---|
2 Abbreviations used in this paper: wt, wild-type; GKO, IFN- knockout; BL/SCID mice, C57BL/6-SCID/SzJ mice; MAg, mycobacterial Ag; KO, knockout; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication September 8, 1997. Accepted for publication January 29, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157:4045.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  S. Fuschillo, A. De Felice, and G. Balzano Mucosal inflammation in idiopathic bronchiectasis: cellular and molecular mechanisms Eur. Respir. J., February 1, 2008; 31(2): 396 - 406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Salvatore, M. Fonseca-Aten, K. Katz-Gaynor, A. M. Gomez, and R. D. Hardy Intranasal Interleukin-12 Therapy Inhibits Mycoplasma pneumoniae Clearance and Sustains Airway Obstruction in Murine Pneumonia Infect. Immun., February 1, 2008; 76(2): 732 - 738. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 S. K. Field and R. L. Cowie Lung Disease Due to the More Common Nontuberculous Mycobacteria Chest, June 1, 2006; 129(6): 1653 - 1672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Pammit, V. N. Budhavarapu, E. K. Raulie, K. E. Klose, J. M. Teale, and B. P. Arulanandam Intranasal Interleukin-12 Treatment Promotes Antimicrobial Clearance and Survival in Pulmonary Francisella tularensis subsp. novicida Infection Antimicrob. Agents Chemother., December 1, 2004; 48(12): 4513 - 4519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Field, D. Fisher, and R. L. Cowie Mycobacterium avium complex Pulmonary Disease in Patients Without HIV Infection Chest, August 1, 2004; 126(2): 566 - 581. [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]
|
|
|
|
|
|
A. Bafica, C. A. Scanga, M. L. Schito, S. Hieny, and A. Sher Cutting Edge: In Vivo Induction of Integrated HIV-1 Expression by Mycobacteria Is Critically Dependent on Toll-Like Receptor 2 J. Immunol., August 1, 2003; 171(3): 1123 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Fieschi, S. Dupuis, E. Catherinot, J. Feinberg, J. Bustamante, A. Breiman, F. Altare, R. Baretto, F. Le Deist, S. Kayal, et al. Low Penetrance, Broad Resistance, and Favorable Outcome of Interleukin 12 Receptor {beta}1 Deficiency: Medical and Immunological Implications J. Exp. Med., February 17, 2003; 197(4): 527 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Greenwell-Wild, N. Vazquez, D. Sim, M. Schito, D. Chatterjee, J. M. Orenstein, and S. M. Wahl Mycobacterium avium Infection and Modulation of Human Macrophage Gene Expression J. Immunol., December 1, 2002; 169(11): 6286 - 6297. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. J. Nau, J. F. L. Richmond, A. Schlesinger, E. G. Jennings, E. S. Lander, and R. A. Young Human macrophage activation programs induced by bacterial pathogens PNAS, January 17, 2002; (2002) 22649799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hesse, M. Modolell, A. C. La Flamme, M. Schito, J. M. Fuentes, A. W. Cheever, E. J. Pearce, and T. A. Wynn Differential Regulation of Nitric Oxide Synthase-2 and Arginase-1 by Type 1/Type 2 Cytokines In Vivo: Granulomatous Pathology Is Shaped by the Pattern of L-Arginine Metabolism J. Immunol., December 1, 2001; 167(11): 6533 - 6544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hayashi, S. P. Rao, K. Takabayashi, J. H. Van Uden, R. S. Kornbluth, S. M. Baird, M. W. Taylor, D. A. Carson, A. Catanzaro, and E. Raz Enhancement of Innate Immunity against Mycobacterium avium Infection by Immunostimulatory DNA Is Mediated by Indoleamine 2,3-Dioxygenase Infect. Immun., October 1, 2001; 69(10): 6156 - 6164. [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]
|
|
|
|
|
|
V. Nagabhushanam and C. Cheers Non-Major Histocompatibility Complex Control of Antibody Isotype and Th1 versus Th2 Cytokines during Experimental Infection of Mice with Mycobacterium avium Infect. Immun., March 1, 2001; 69(3): 1708 - 1713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mohagheghpour, A. van Vollenhoven, J. Goodman, and L. E. Bermudez Interaction of Mycobacterium avium with Human Monocyte-Derived Dendritic Cells Infect. Immun., October 1, 2000; 68(10): 5824 - 5829. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Doherty, C. Chougnet, M. Schito, B. K. Patterson, C. Fox, G. M. Shearer, G. Englund, and A. Sher Infection of HIV-1 Transgenic Mice with Mycobacterium avium Induces the Expression of Infectious Virus Selectively from a Mac-1-Positive Host Cell Population J. Immunol., August 1, 1999; 163(3): 1506 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shimizu, H. Tomioka, K. Sato, C. Sano, T. Akaki, S. Dekio, Y. Yamada, T. Kamei, H. Shibata, and N. Higashi Effects of the Chinese Traditional Medicine Mao-Bushi-Saishin-To on Therapeutic Efficacy of a New Benzoxazinorifamycin, KRM-1648, against Mycobacterium avium Infection in Mice Antimicrob. Agents Chemother., March 1, 1999; 43(3): 514 - 519. [Abstract] [Full Text]
|
|
|
|
|
|
K. Mohan, H. Sam, and M. M. Stevenson Therapy with a Combination of Low Doses of Interleukin 12 and Chloroquine Completely Cures Blood-Stage Malaria, Prevents Severe Anemia, and Induces Immunity to Reinfection Infect. Immun., February 1, 1999; 67(2): 513 - 519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Silva, T. F. Pais, and R. Appelberg Evaluation of IL-12 in Immunotherapy and Vaccine Design in Experimental Mycobacterium avium Infections J. Immunol., November 15, 1998; 161(10): 5578 - 5585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Nau, J. F. L. Richmond, A. Schlesinger, E. G. Jennings, E. S. Lander, and R. A. Young Human macrophage activation programs induced by bacterial pathogens PNAS, February 5, 2002; 99(3): 1503 - 1508. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
were
significantly different from mice treated with drug and cytokine
together. A single marker indicates a difference that was significant
at the p < 0.05 level, while a double marker indicates
a significant difference at p < 0.001. ND = not
done. The results shown are typical of three experiments
performed.
