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Transgene1
Department of Pathology and Molecular Medicine, and Division of Infectious Diseases, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-12, but not TNF-
, during M. bovis bacillus
Calmette-Guérin infection. These mice succumbed to such
infection. A repeated lung gene transfer strategy was designed to
reconstitute IFN- in the lung, which allowed investigation of
whether adequate activation of innate macrophages could enhance host
defense in the complete absence of lymphocytes. IFN- transgene-based
treatment was initiated 10 days after the establishment of
mycobacterial infection and led to increased levels of both IFN- and
IL-12, but not TNF-, in the lung. Lung macrophages were activated to
express increased MHC molecules, type 1 cytokines and NO, and increased
phagocytic and mycobactericidal activities. Activation of innate
immunity markedly inhibited otherwise uncontrollable growth of
mycobacteria and prolonged the survival of infected SCID hosts. Thus,
our study proposes a cytokine transgene-based therapeutic modality to
enhance host defense in immune-compromised hosts against intracellular
bacterial infection, and suggests a central effector activity played by
IFN--activated macrophages in antimycobacterial cell-mediated
immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- (1, 2, 3). In
vitro studies suggest that macrophages activated by these cytokines
acquire an increased ability to control the replication of
intracellular mycobacteria (3). In addition to activated
macrophages, however, both CD4/CD8 T cells and NK cells are also
capable of antimycobacterial effector activities by directly
killing infected macrophages, inhibiting mycobacterial replication, and
lysing mycobacteria (3, 4, 5, 6). Thus, the issue regarding to
which extent activated macrophages by themselves are able to control
mycobacterial infection in vivo has remained to be understood.
Humans with such immune-compromised conditions as SCID, AIDS, and
genetic deficiencies in type 1 cytokines IFN- and IL-12 or their
receptors are especially susceptible to intracellular infection by
normally otherwise nonpathogenic strains of mycobacteria such as
Mycobacterium bovis bacillus Calmette-Guérin
(BCG)3 (1, 2, 3, 7, 8). It is known that chemotherapeutic modalities are often
ineffective in these hosts without the aid provided by key intrinsic
immune components. Future effective therapy will most likely involve
the use of immunotherapeutics aiming to enhance the function of
remaining immune components such as macrophages in these hosts.
However, the rationale and feasibility of such strategy still remain to
be established. Apparently, the development of this strategy rests on
the important definition of the ability of macrophages to control
mycobacterial infection in vivo, independent of cytotoxic T and NK
cells.
Since the lung is naturally much more prone to mycobacterial infection than other tissue sites (9, 10), we employed a SCID beige mouse model of pulmonary M. bovis BCG infection that we have previously established (11) to investigate several questions of fundamental importance: 1) whether SCID hosts have an intact tissue macrophage population available for immune activation; 2) whether SCID hosts have impaired type 1 cytokine responses during myocbacterial infection; 3) whether a cytokine transgene-based therapeutic modality aiming to activate innate macrophages, when given after the establishment of infection, is able to enhance the control of mycobacterial replication in the absence of both lymphocytes and NK cells; and 4) if so, what are the mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C.B-17 SCID beige mice were bred in our central animal facility under Level A specific pathogen-free housing conditions until use. Immune-competent BALB/c and C57BL/6 mice (Harlan, Indianapolis, IN) were housed under Level B specific pathogen-free housing conditions until use. After infection, all mice were kept in autoclaved cages with autoclaved bedding, food, water, and microfilter lids in a biohazard Level B facility. All experiments performed were in accordance with the guidelines of the Animal Research Ethics Board of McMaster University.
Infectious agent and viral cytokine gene transfer vectors
Live M. bovis BCG was originally obtained from Connaught Laboratories (North York, Ontario, Canada). It was grown in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with Middlebrook OADC enrichment (Life Technologies, Gaithersburg, MD), 0.002% glycerol, and 0.05% Tween 80. A replication-deficient adenoviral vector was used for cytokine gene transfer in vivo. This virus has its E1 and E3 genomic regions partially deleted, but remains fully infectious (12). As control, a replication-deficient adenoviral vector that does not contain cytokine transgene was used. All adenoviral gene vectors were amplified, purified, and titrated in our Center by using the standard procedures previously described by us (12).
Pulmonary mycobacterial infection and intranasal cytokine gene transfer
Pulmonary mycobacterial infection was established via the
airway, as previously described (11, 13, 14). Before
infection, BCG stock solution was diluted in PBS, and the preparation
was sonicated to ensure proper dispersion of mycobacteria. Mice were
infected by intratracheal instillation of live BCG at a dose of 5
x 105 CFU in a total volume of 40 µl/mouse, as
previously described. Recombinant replication-deficient adenoviral
vector expressing murine IFN- (15) (kindly provided by
J. Kolls at Louisiana State University, New Orleans, LA) or murine
GM-CSF (16) or the control vector (Add170) was diluted
into 4 x 108 PFU in a total volume of 30
µl in PBS. Intrapulmonary cytokine gene transfer was conducted by
intranasal (i.n.) delivery of adenoviral vector, as previously
described (16). Briefly, half of 30 µl virus suspension
was applied to the mouse nasal orifice so that the mouse could breathe
in the suspension naturally. The second half was applied in the same
way thereafter. We have previously demonstrated that after i.n. gene
transfer, transgene was expressed primarily by airway epithelial cells
for a period of 10–12 days (16, 17, 18).
Bronchoalveolar lavage, lung histology, and lung macrophages isolation
At selected time points postintrapulmonary gene transfer, groups of mice were sacrificed and lungs were removed and subjected to bronchoalveolar lavage (BAL), followed by perfusion with 10% Formalin for histology. BAL was conducted by following a standard procedure previously described (11, 13). Briefly, to collect BAL fluid, a polyethylene tube (Becton Dickinson, Sparks, MD) was used to cannulate the trachea. Lungs were lavaged twice with PBS. BAL samples were spun at 4000 rpm for 2 min at 4°C, and supernatants were removed and stored at -20°C until cytokine assay. Lungs were fixed in 10% Formalin by perfusion. Both left and right lungs were sectioned from top to bottom, resulting in four to five cross-sectional pieces of tissue from each side. Tissues were then embedded in paraffin, cut into 4- to 5-µm sections, and stained with H&E or Ziehl-Neelsen for identification of mycobacteria.
Following BAL, alveolar macrophages were collected. For each experiment, macrophages isolated from three to five mice were pooled. These cells were resuspended in RPMI culture media containing 10% FBS and cultured for 3 days in 96-well plates at a density of 0.1 x 106 cells/well without or with various stimuli. The supernatants were collected and stored at -20°C until cytokine assay.
Measurement of cytokines in BAL and macrophage culture supernatants
Cytokines were measured in BAL and cell culture supernatants by
specific ELISA. All ELISA kits were purchased from either R&D Systems
(Minneapolis, MN) (IFN- and TNF-) or Biosource (Montréal,
Québec, Canada) (IL-12). The sensitivity of detection for all of
these ELISA kits was 
5–10 pg/ml.
Mycobacterial colony enumeration assay
A colony assay was performed as previously described (13). Briefly, lungs were placed in 4.5 ml PBS/0.05% Tween 80, cut into small pieces (2–3 mm) under sterile conditions, and homogenized with a tissue homogenizer. Homogenates were allowed to settle on ice for 30 min, and properly diluted homogenates were plated onto each plate of Middlebrook 7H10 agar containing OADC enrichment (Difco). Plates were incubated at 37°C, and colonies were counted using a dissecting microscope at days 11 to13.
Measurement of NO production
The release of NO by lung macrophages was determined by measuring the concentration in culture supernatants of the end product of NO, nitrite, as previously described (19). Briefly, diluted supernatants were mixed at a 1:1 ratio with Griess reagent buffer (Sigma, St. Louis, MO). The absorbance was determined at 540 nm by using a spectrophotometer. The final concentrations of nitrite were calculated from a standard curve derived from prepared solutions of NaNO2 of known concentrations.
Evaluation of macrophage activation by FACS analysis
Immunostaining and FACS analysis procedures were conducted as previously described (18, 20). Briefly, isolated total leukocytes from SCID mouse lung were incubated with anti-FcR Ab 2.4G2 on ice for 15 min and then stained with FITC-labeled anti-MHC class I or biotinylated anti-MHC class II Abs on ice for 30 min. After wash with PBS/0.2% BSA, the cells were labeled with PE-conjugated anti-mouse CD11b (Mac-1) Ab for 30 min on ice. Data were collected from FACScan (Becton Dickinson, Sunnyvale, CA). Analysis was performed by gating on the macrophage-rich area, excluding lymphocytes and dead cells. Since this area might also contain some granulocytes, the results from FACS analysis were interpreted by taking into account the percentage of macrophages in total BAL-derived leukocytes determined by differential staining of cytospins of BAL cells before FACS staining.
Macrophage phagocytosis and mycobactericidal assays
Macrophages were isolated from the lung of naive SCID mice
receiving the control or IFN- gene transfer vector. Cells were
allowed to interact with live BCG bacilli (1:10 macrophage:bacilli) for
2 h at 37°C in 24-well plates at a density of 0.2 x
106 cells/well in RPMI media containing 5% FBS
without antibiotics. Free, nonphagocytosed bacilli were removed by
rinsing the monolayer of macrophages three to four times with culture
media. Macrophages were then removed from the plate, and cell cytospins
were made. Cytospins were stained with cold KINYOUN stain for acid-fast
bacilli. At least 200–300 cells were counted on each cytospin. Both
the number of intracellular bacilli in each macrophage and the number
of macrophages that phagocytosed bacilli were recorded. To determine
the rate of intracellular mycobacterial replication, macrophages were
cultured with live BCG bacilli, as above. At both days 0 and 3, 0.5 ml
cold 0.25% SDS was introduced into the plate to lyse macrophages. The
number of intracellular bacilli was compared between day 0 and day 3 by
a colony enumeration assay.
Data analysis
Whenever applicable, the difference comparison was made by using
a Student t test. The difference was considered
statistically significant when p 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that SCID beige hosts are unable to
control pulmonary mycobacterial infection by M. bovis BCG
bacilli as a result of the lack of both lymphocytic repertoire and NK
cells (11). While these mice were found to have an
impaired ability to form well-organized granuloma in their lungs,
particularly at earlier stages of infection, the level of macrophage
infiltration in the lung appeared normal (11). To
determine whether there were any defects in macrophage functions, we
compared both the number and function of alveolar macrophages between
naive SCID beige and immune-competent control mice. As shown in Table I
, SCID mice contained a similar number
of macrophages compared with wild-type mice. Furthermore, these cells
responded as well as those from control mice to GM-CSF stimulation in
vitro in undergoing proliferation. Upon LPS stimulation or
mycobacterial infection, SCID macrophages released similar amounts of
TNF-. These findings indicate that regardless of the lack of
lymphocytes and NK cells, SCID beige mice have a normal macrophage
population. This strain of mice thus provided us a unique tool to
investigate the role of macrophage activation in host defense against
mycobacterial infection in the absence of adaptive immune
components.
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To identify the cytokine that may be missing during
antimycobacterial immune responses in SCID beige mice and thus could be
chosen as a candidate for replacement to activate macrophages, we
analyzed the level of type 1 cytokines IFN-, IL-12, and TNF- in
the lung of SCID mice at day 37 postpulmonary mycobacterial infection.
Compared with immune-competent mice, SCID mice had much less IFN-
and IL-12 in the lung, whereas the level of TNF- was not diminished
(Fig. 1). Since T cells, NK cells, and
macrophages can all be a source of IFN- (21, 22),
diminished IFN- responses most likely resulted from the lack of T
and NK cells as well as the lack of macrophage activation. The
unimpaired TNF- response suggests that this cytokine was primarily
released from macrophages, since we have recently shown that
mycobacteria could directly release TNF- from macrophages,
independent of IL-12, in contrast to macrophage IFN- release
(19, 20), and that this cytokine by itself was unable to
adequately activate macrophages to enhance their mycobactericidal
activities. Thus, markedly weakened host defense against mycobacterial
infection in SCID hosts is most likely attributable to inadequate
responses of IFN- and IL-12 and the subsequent lack of macrophage
activation. Since IFN- is a potent macrophage activator critically
required for antimycobacterial host defense (20, 23, 24),
we chose to reconstitute the level of this cytokine in the lung of SCID
beige mice by using an intrapulmonary gene transfer approach.
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gene transfer to the lung of SCID mice
Our previous studies have demonstrated that intrapulmonary
adenoviral gene transfer leads to raised levels of cytokine product in
the lung for a period of 10 to 12 days (16, 17, 18). Since
pulmonary mycobacterial infection is a chronic process and we have
demonstrated a sustained IFN- response present in the lung of
immune-competent hosts during pulmonary mycobacterial infection
(11, 13), repeated IFN- gene transfer to the lung may
be required to sustain the level of IFN- and to significantly
enhance host defense in SCID mice. The lack of adaptive immune
components in SCID mice would allow the efficient repeated
adenoviral-mediated gene transfer. To establish the feasibility of such
repeated gene transfer in SCID hosts, we delivered the first dose
(4 x 108 PFU) of adenoviral IFN- gene
transfer vector, i.n. to naive SCID mice, and examined the level of
IFN- protein in the BAL fluid at days 4 and 14. Single gene transfer
resulted in markedly increased levels of IFN- by day 4 (2000 pg/ml),
which declined to 
40 pg/ml by day 14 (Fig. 2A). However, if a second dose
of IFN- vector was given to such mice at day 10, the level of
IFN- rose back to 2000 pg/ml again at day 14 (Fig. 2A). These findings thus indicate the feasibility of
repeated IFN- gene transfer for reconstituting and sustaining
IFN- expression in the lung of SCID mice, to a level similar to that
in the lung of immune-competent mice during pulmonary mycobacterial
infection (13).
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gene transfer to SCID mice
To examine whether IFN- gene transfer modality could
effectively activate macrophages in SCID beige mouse lung in a
clinically relevant setting, pulmonary mycobacterial infection was
established first in SCID mice, and then the initial dose of adenoviral
IFN- gene transfer vector was delivered i.n. to these mice 10 days
after the establishment of infection. A second dose of IFN- gene
vector was given at day 20 postinfection to prolong the level of
IFN- (Fig. 2
B, experimental regimen). Mice were
sacrificed at days 27, 37, and 52 postinfection, and the level of
infection was assessed by a colony enumeration assay.
SCID mice that were treated with a control vector failed to control
infection and demonstrated uncontrollable, escalating replication of
mycobacteria in the tissue (Fig. 3). In
contrast, mice that were treated with IFN- transgene demonstrated at
least 10-fold lower bacterial counts at days 27 and 37, and 7.7-fold
lower bacterial counts at day 52. Histologically, while loads of
bacilli were easily located within macrophages in control mouse lung,
much fewer bacilli were seen in the lung of mice treated with IFN-
transgene (Fig. 4, A and
B). Of note, IFN- transgene treatment only moderately
increased the number of macrophages/monocytes in the lung (not shown)
and did not markedly improve macrophage granuloma formation in the
lung, in contrast to a vigorous granulomatous response seen in the lung
of immune-competent mice (Fig. 4, C–E). These findings
suggest that IFN- transgene treatment improved host resistance
primarily via its effect on the quality of macrophages in the lung of
infected SCID mice.
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deliveries could prolong the survival of
infected SCID mice. We have previously observed that untreated SCID
mice will succumb to M. bovis BCG infection
(11). Thus, we infected and treated SCID mice with the
control vector or IFN- gene vector (8–10 mice/group), as described
in Fig. 2B, except that after the second dose of vector,
additional three doses of vector were given, 10 days apart, to
prolong the level of IFN- in the lung. The mortality of mice was
monitored. We found that mice in control group became morbid around day
40 and started to die around day 50, and all mice died by day 80 with
an average survival of approximately 60 days (Fig. 5). In comparison, mice treated with
IFN- transgene appeared healthy at earlier stages and did not start
to die until 70 days, and 25% of mice survived beyond 90 days with
a significantly prolonged survival rate (Fig. 5).
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and IL-12, but not TNF-, in the lung
by IFN- gene transfer
To investigate the mechanisms by which IFN- transgene enhanced
protection against mycobacterial infection in SCID beige mice, we
examined the cytokine profile in the lung. The concentration of type 1
cytokines IFN-, IL-12, and TNF- in the BAL fluid was determined 7
days post-second IFN- gene delivery (day 27 postinfection) (Fig. 2B). Following IFN- gene transfer, a significant amount
of IFN- was measured in the lung, whereas it was barely detectable
in the lung of control mice at this time point (Fig. 6A). The level of IL-12 was
also elevated in the lung of IFN- transgene-treated mice (Fig. 6B). Of interest, the level of TNF- was similar in both
control and IFN- transgene-treated SCID mice (Fig. 6C),
suggesting that IFN- transgene expression selectively up-regulated
the release of IL-12, but not TNF-, in this model.
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gene transfer in
the lung
We next examined whether heightened type 1 cytokines enhanced
antimycobacterial host defense by activating macrophages. To this end,
we first examined the phenotype of lung macrophages isolated 7 days
post-second gene transfer (27 days postinfection) from SCID mice for
their surface expression of CD11b and MHC class I and II molecules.
IFN- is a potent inducer of MHC class I and II molecules.
Macrophages express high density of CD11b and MHC class I and, when
activated, MHC class II molecules (20). In this particular
experiment, an adenoviral vector expressing murine GM-CSF was used as a
control to compare with the vector expressing IFN-. By a cytologic
analysis conducted before FACS staining, we found that the majority of
cells to be examined by FACS analysis were macrophages (95%, 80%, and
91% for adenoviral E1-deleted l70-3, adenoviral GM-CSF, and adenoviral
IFN-, respectively). Approximately 20% of cells from control
vector-treated SCID mice expressed bright CD11b and MHC class I (Fig. 7, top panels). Expression of
GM-CSF in the lung only slightly increased the size of this cell
population (25%). In contrast, IFN- gene transfer markedly
increased this cell population expressing bright CD11b/MHC class I to
48%. A similar picture was seen with MHC class II staining (Fig. 7, bottom panels). While the number of macrophages expressing
bright CD11b/MHC II was small and similar in both control and GM-CSF
groups (1–2%), IFN- transgene treatment increased this macrophage
population to 9%. These findings suggest that compared with other
macrophage stimuli such as GM-CSF, IFN- is a uniquely potent
macrophage activator during mycobacterial infection.
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gene
transfer in the lung
To investigate whether lung macrophages phenotypically activated
by IFN- gene transfer released increased amounts of soluble
mediators critical to antimycobacterial host defense, intraalveolar
macrophages were isolated 7 days post-second i.n. gene transfer (day 27
postinfection) and cultured in vitro under various conditions. The
release of both type 1 cytokines and NO was determined. We found that
in contrast to those from control mice, macrophages from IFN-
transgene-treated mice spontaneously released large amounts of IFN-
(441 pg/ml vs 22 pg/ml in controls) and IL-12 (257 pg/ml vs 54 pg/ml in
controls) (Fig. 8, A and
B). Upon stimulation with LPS, these cells released further
increased amounts of these cytokines. In comparison, macrophages from
both control and IFN- transgene-treated mice released comparable
amounts of TNF- spontaneously or upon LPS stimulation (Fig. 8C). These results suggest that cytokines measured in the
lung were released primarily from activated macrophages since the
profile of cytokine production in alveolar macrophages mirrors that in
the lung (Fig. 6).
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is a key inducer of NO in
macrophages during mycobacterial infection and NO is a potent
mycobactericidal chemical compound required for host defense (19, 25, 26), we further examined the ability of macrophages to
release NO ex vivo. Macrophages from IFN- transgene-treated mice
spontaneously released a much greater amount of NO than those from
controls (Fig. 9, A and
B). Stimulation with LPS, IFN-, or IL-12 further enhanced
the release of NO. These findings suggest that IFN- transgene
treatment in SCID mice enhanced host defense against mycobacterial
infection via its effect on macrophage activation, particularly NO
release.
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gene transfer in the lung
To examine whether activation of macrophages by IFN- gene
transfer to the lung translated to increased antimycobacterial
activities in macrophages, macrophages were isolated from the lung of
SCID beige mice that were treated i.n. with control vector or IFN-
gene transfer vector without mycobacterial infection. These cells were
then allowed to interact with live mycobacteria in vitro, and their
ability to phagocytose and to inhibit the growth of intracellular
mycobacteria was analyzed. By using a phagocytosis assay, a greater
number of macrophages from IFN--treated mice were found to have
engulfed mycobacteria (Table II).
Furthermore, the average number of engulfed bacilli per macrophage was
also greater in macrophages of IFN- transgene group (3.41/macrophage
vs 1.81/macrophage in control group).
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-activated macrophages was approximately 3 times greater than
that from control macrophages (Table II), again indicating that
IFN--activated macrophages more avidly phagocytosed mycobacteria. By
day 3, while the number of intracellular bacilli in control macrophages
increased by 141%, the number of bacilli in IFN--activated
macrophages decreased by 11% (Table II). These findings indicate
that IFN- gene transfer-mediated macrophage activation endows
macrophages with a greater ability to engulf mycobacteria and control
their replication. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12, but not TNF-, responses; 3) repeated IFN- gene
transfer to SCID mouse lung, after the establishment of mycobacterial
infection, enhanced host defense in the absence of lymphocytes and NK
cells; and 4) IFN- gene transfer enhanced host defense in SCID
hosts, not through improving macrophage granuloma formation, but rather
by activating macrophages. These observations demonstrate the rationale
and feasibility to control the otherwise uncontrollable mycobacterial
infection in immune-deficient humans by tailoring the innate immunity
with macrophage-activating transgene-based therapeutics. To our
knowledge, our study represents the first to demonstrate that IFN-
transgene-based therapeutic is effective in enhancing host defense
against a fatal intracellular bacterial infection in the absence of T
and B lymphocytes and NK cells.
Mycobacteria are facultative intracellular pathogens of macrophages.
Increasing experimental evidence suggests that mycobacteria evade the
intracellular bactericidal mechanisms of macrophages by interfering
with the process of phagosome-lysosome fusion and the acidification of
phagolysomal compartment (27). The ultimate control of
mycobacterial replication and infection has been believed to involve
several mechanisms of cell-mediated immunity (1, 2, 3, 4, 5, 6, 25, 26, 27, 28): 1) activation of infected macrophages by T and NK
cell-derived cytokines, including IFN- and TNF-, and increased
mycobactericidal activities in these activated macrophages; and 2)
inhibition of mycobacterial replication and direct lysis of infected
macrophages and mycobacteria by cytotoxic CD4 and CD8 T and NK cells
via perforin/granzyme-, Fas/FasL-, and granulysin-mediated mechanisms.
It has thus remained difficult to evaluate the relative contribution by
macrophages themselves to the control of intracellular infection.
Although RAG-1-/- and conventional SCID mouse
strains are useful for addressing the role of T and B cells, the
presence of NK cells in these mouse strains precludes a definitive
conclusion about the contribution by macrophages. It has been found
that these mice could provide certain protection, via NK cell-derived
IFN- and cytotoxic activities, against intracellular infection by
listeria, Mycobacterium avium, Cryptosporidium parvum, and
Shigella flexner (21, 29, 30, 31). By using a model
of mycobacterial infection in SCID beige hosts deficient in both
lymphocytes and NK cells, in conjunction with the use of an IFN-
gene transfer vector, we had a unique opportunity to investigate the
role of macrophages, before and after activation, in host defense
against mycobacterial infection.
We have previously found that compared with immune competent mice and
mice with a selective defect in an immune component (IL-12, CD4, or CD8
T cells), SCID beige mice are the most susceptible to pulmonary
mycobacterial infection by M. bovis BCG (11, 13, 14). These mice demonstrated diminished granuloma formation and
progressively increased bacillary load in the tissue. In our current
study, the level of IFN- and IL-12 was found to be weakened, and in
comparison, the level of TNF- was unimpaired both in the lung and
macrophages. Since these mice have a functionally normal macrophage
population, as shown in our current study, the impaired granuloma
formation cannot be attributed to any aberrant function of macrophages.
Nor was it due to the lack of TNF-, although this cytokine was shown
to be required for granuloma formation during mycobacterial infection
(32, 33, 34). The unimpaired TNF- response to mycobacterial
infection in the absence of T and NK cells suggests that first of all,
this cytokine is macrophage derived, and second, this cytokine on its
own is insufficient for granuloma formation and macrophage activation.
Granuloma formation most likely requires both TNF- and the presence
of T cells. Indeed, macrophage TNF- release in response to
mycobacterial infection is independent of both IL-12 and IFN-
(19, 20). Markedly decreased IL-12 production concurrent
with decreased IFN- was somewhat unexpected in SCID beige mice, but
is nonetheless in agreement with the notion that the optimal IL-12
release requires both IFN- and intracellular pathogen (35, 36), a T cell-independent IL-12 release mechanism. T
cell-dependent IL-12 release mechanism is apparently voided in SCID
mice. The lack of IFN- from both NK cells and T cells in SCID beige
hosts accounts for the weakened IL-12 production. All of these findings
indicate that when minimally activated in SCID beige hosts, macrophages
are unable to control mycobacterial infection, and such inability is
associated most closely with the lack of IFN-, but not TNF-.
Thus, to investigate whether macrophages in SCID beige hosts could be
adequately activated to combat mycobacterial infection, an adenoviral
gene transfer vector was chosen to express IFN- in the lung. Such
gene vector targets the transgene to airway epithelial cells and leads
to a prolonged, raised level of transgene product in the lung of
immune-competent mice (16, 17, 18). We have shown in the
current study that the lack of adaptive immune components in SCID mice
allows an even more sustained level of transgene product by allowing
repeated gene transfer in the lung. Thus, we would be able to create an
IFN--rich environment similar to that in an immune-competent host
during mycobacterial infection (13). This strategy shall
lead to the most efficient activation of macrophages. IFN- transgene
expression rendered after the establishment of infection led to
heightened levels of IFN- in the lung. As a result, the level of
IL-12 was increased, reinforcing the notion that the optimal production
of IL-12 requires IFN- in a lymphocyte/NK cell-deficient host. Of
importance, increased IFN- was not accompanied by increased TNF-
nor by significantly improved granuloma formation, which again suggests
that granuloma formation requires both type 1 cytokines and T cells.
Airway expression of IFN- transgene is remarkably effective in
activating macrophages. Macrophages were activated to release increased
amounts of not only IFN-, but IL-12 as well as NO. They also
expressed phenotypic activation markers, including MHC class I and
class II molecules. These changes in macrophage activation status
translated to increased antimycobacterial activities. These activated
macrophage properties induced by IFN- gene transfer in SCID mouse
lung highly resemble those found in immune-competent mouse lung
(20). The coupling of increased IFN- release with
increased IL-12 both in the lung and macrophages suggests that not only
the optimal production of IL-12 requires IFN- in SCID mice, but
IL-12 may in turn release more IFN- from macrophages since we have
shown that macrophage IFN- release requires two signals:
mycobacteria and IL-12 (19, 20). Indeed, we found that
activated macrophages spontaneously released increased amounts of both
IFN- and IL-12 ex vivo.
Hence, IFN- transgene-mediated macrophage activation clearly led to
enhanced control of mycobacterial replication in the absence of CD4 and
CD8 T and NK cells, at least within the time frame of our current
study. It is of importance to note that mycobacterial infection in SCID
beige mice by attenuated M. bovis BCG is a very chronic
process, different from that by virulent Mycobacterium
tuberculosis, and the most effective control of infection may
require a much more prolonged regimen with IFN- transgene. In the
mortality study presented in the current study, however, the IFN-
transgene regimen consisted of only five repeated doses starting from
day 10 postinfection, 10 days apart thereafter, and a significant
number of mice became moribund around day 80, a time when IFN-
transgene product from the last dosing tapered off. This very likely
disallowed the persistence of enhanced immune protection and conferred
only a limited prolonged survival in these IFN- transgene-treated
SCID mice. Thus, we expect that if the IFN- transgene treatment
continued beyond five repeated doses, these mice may experience a
long-term survival. On one hand, our findings suggest that macrophages
are a central effector cell in host defense against mycobacterial
infection, and that the primary role played by T and NK cells in
cell-mediated immunity is to warrant adequate macrophage activation by
providing the key type 1 cytokine IFN-. Recent studies with perforin
or granzyme gene knockout mice seem to lend further support to this
conclusion (37, 38). On the other hand, however, our
findings also suggest that IFN--mediated macrophage activation
alone, in the absence of T cells, may not be the entire answer to the
eradication of intracellular mycobacteria (infection rebounds as
IFN- levels decline). The fact that prolonged IFN- transgene
expression in the lung of SCID beige mice, in the presence of
endogenous TNF-, fails to enhance granuloma formation suggests that
T cells assist in granuloma formation, which, together with IFN-,
might be required for effective control of mycobacterial infection.
This notion does not seem to support the conclusion from a recent study
by Johnson and colleagues (39) that significant immunity
to M. tuberculosis is not ablated in the absence of
granuloma formation in ICAM-1-deficient mice.
Severely immune-compromised humans are susceptible to infection by
normally otherwise nonpathogenic intracellular bacteria, particularly
M. bovis BCG and environmentally borne mycobacterial
strains. Although rIFN- protein has been shown to provide certain
therapeutic benefits to immune-competent hosts with M.
tuberculosis or Mycobacterium leprae infection
(40, 41), it will unlikely be effective in control
mycobacterial infection in severely immune deficient hosts. Indeed, by
systemic delivery of rIFN- protein, Flynn and colleagues
(23) could only minimally prolong the survival of
IFN--deficient mice with M. tuberculosis
infection. Our current study thus supports the local use of IFN-
transgene-based formulations to turn the innate immunity around in
humans that are deficient in T cells or IL-12 or IL-12Rs (42, 43). Although still remained to be demonstrated, such IFN-
transgene therapeutic modality is expected to be even much more
effective when used in conjunction with antimycobacterial
chemotherapy.
|
Acknowledgments |
|---|
vector,
Xueya Feng and Duncan Chong for amplification and purification of
adenoviral vectors, and Michael Santosuosso for assistance in NO
assay. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Zhou Xing, Room 4H19, Department of Pathology and Molecular Medicine, Health Sciences Center, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5 Canada. E-mail address: xingz{at}mcmaster.ca
3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; Ad, •••; Addl, •••; BAL, bronchoalveolar lavage; i.n., intranasal.
Received for publication February 28, 2001. Accepted for publication April 27, 2001.
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References |
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