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and Primed Lymphocytes
Cornell University Medical College, Division of International Medicine and Infectious Diseases, Department of Medicine, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Omission of IFN- in the cocultures or Mtb
lysate/IFN--primed lymphocytes was associated with high growth of
Mtb, high IL-10 and IL-12 p40, nearly undetectable IL-12
p70 levels, and the highest percentages of CD4 and CD8 T cells. In
contrast, IFN- treatment of cocultures containing Mtb
lysate/IFN--primed PBL reduced bacilli count by 
2.5 log,
decreased the production of IL-10 by 5.7-fold, increased IL-12 p70 by
50-fold, and reduced the percentages of CD4 and CD8 T cells.
Activation of anti-Mtb activity was time and dose
dependent. At 2000 U/ml of IFN-, bactericidal activity was achieved
(10-fold reduction from initial inoculum). Anti-Mtb
activity against several strains of M. tuberculosis (H37Ra
and H37Rv, and C, a clinical isolate) was observed and was associated
with expression of inducible nitric oxide synthase. These data suggest
that induction of human macrophage anti-Mtb activity
required dual signaling from PBL and IFN-. Thus, the development of
an in vitro human system may greatly facilitate studies to delineate
immune cells, cytokines, and effector functions/genes critical in
controlling Mtb. Defining the mechanisms may also provide
novel treatment strategies for tuberculosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Little is known of the early interactions of microbes and immune cells that result in either restricted infection or dissemination and disease, nor of the reasons why some individuals reactivate latent infection. However, cell-mediated immunity is critical for restricting Mtb infection; this is highlighted by the increased risk of tuberculosis associated with decreased cellular immunity such as by immunosuppressive drugs, certain cancers, and the acquired immunodeficiency syndrome. Anti-tuberculous cellular immunity involves the critical interplay of T lymphocytes, macrophages, and cytokines (3, 4). Mycobacterium-specific CD4 and CD8 T lymphocytes have been identified that have cytolytic activity against mycobacteria-harboring macrophages (4, 5, 6, 7, 8). In mice infected with Mtb complex, depletion of CD4 T lymphocytes results in disseminated disease, while competent mice have restricted infection (4, 5). Similarly, when CD4 T cell counts decrease in HIV-1-infected persons, the risk of tuberculosis is increased whether from primary infection or from reactivation of latent Mtb infection (9). In contrast to CD4 T lymphocytes, the role of Mtb- or Mycobacterium avium-specific CD8 T cells in experimental murine infection and in humans remains undefined (3, 4).
In vitro human and murine Mycobacterium Ag-specific CD4 T
cells produce plentiful amounts of IFN- (7, 8). The production of
IFN- by CD4+ T cells is thought to activate macrophages
to control intracellular microbes (3, 5, 7, 8, 9, 10, 11, 12). Homologous knockout of
IFN- ligand or receptor genes in mice results in defective
macrophage antimicrobial effector function, as exemplified by impaired
expression of inducible nitric oxide synthase (iNOS, NOS2), MHC class
II Ag, and disseminated mycobacterial infection (13, 14, 15). In man,
mutations of the IFN- receptor rendering it functionless are
associated with fulminant tuberculosis and disseminated mycobacteremia
after vaccination with Mycobacterium bovis bacillus
Calmette-Guérin (BCG) (16, 17). Although induction of in vitro
murine macrophage anti-Mtb activity by IFN- has been
observed, whether human macrophages can achieve anti-Mtb
activity in vitro via induction by IFN- remains controversial
(18, 19).
The purpose of this study is to develop an in vitro system to delineate
the host modulators, cellular elements, and effector functions critical
for control of Mtb infection. We report the induction of in
vitro human macrophage anti-Mtb activity and the
requirement for coculture with autologous lymphocytes primed with
IFN- plus Mtb Ag and additional treatment with IFN-.
Studies using this model system may facilitate the characterization and
delineation of host and microbial factors critical for controlling
Mtb.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The materials and reagents used and the respective manufacturers
are as follows: RPMI 1640, gentamicin sulfate, M199 medium, and
L-glutamine (BioWhittaker, Walkersville, MD); penicillin,
streptomycin, and HEPES (Life Technologies, Grand Island, NY); Triton
X-100 (Sigma Chemical, St. Louis, MO); normal saline (Baxter Health
Care, Deerfield, IL); BSA (Boehringer Mannheim Biochemicals,
Indianapolis, IN); purified protein derivative (PPD) of Mtb,
Pasteur-Merieux-Connaught, Ontario, Canada); 7H9 broth, Tween-80, and
Middlebrook 7H11 agar (Difco, Detroit, MI); 24-well flat-bottom plates
and 96-well round-bottom plates (Corning Glass, Corning NY); 13-mm
diameter Gold Seal glass coverslips (Clay Adams/Becton Dickinson
Labware, Lincoln Park, NJ); IFN- (Genentech, San Francisco, CA);
Kinyoun Carbol Fuchsin (Becton Dickinson, Cockeysville, MD);
[3H]thymidine (New England Nuclear, Boston, MA); IL-12
p40 EIA kit (R&D Systems, Minneapolis, MN); IL-10 and IL-12 p70 EIA
kits (Immunotech, Coulter, Hialiah, FL); phycoerythrin-labeled
anti-CD3 and FITC-labeled anti-CD4 and anti-CD8 mAbs
(Coulter); CCL64 mink lung fibroblasts (Mv1Lu; American Type Culture
Collection (ATCC), Bethesda, MD); and Live/Dead Viability/Cytotoxicity
Kit (Molecular Probes, Eugene, OR). To remove endotoxin, tissue culture
medium (composed of RPMI 1640, 100 U/ml penicillin, 100 g/ml
streptomycin, 5 mM HEPES, and 5 mM L-glutamine) was
ultrafiltered through Hemo-Flow F-40 (MTS Medizin-Technischl System,
Fresensius, Germany) and is herein designated as ultrafiltered medium
(UFM).
Enumeration of mycobacteria and preparation of mycobacterial Ag
Mtb strains H37Ra and H37Rv (ATCC nos. 25177 and 27294, respectively) and a New York City clinical isolate, designated as the C strain (20), were grown in 7H9 broth supplemented with 0.05% Tween-80. Log phase cultures were pelleted, washed three times in RPMI 1640, and resuspended in RPMI 1640. The cell suspension was homogenized in glass beads by vortexing (3 min), and aggregates were allowed to sediment at 1 x g for 3 to 5 min. A bacterial suspension (in some instances in 20% glycerol) was aliquoted and stored at -70°C. The number of viable mycobacteria after storage was estimated by CFU by serial dilutions in normal saline (NS) and growth on Middlebrook 7H11 agar in tape-sealed plates at 37°C in 5% CO2. CFU were simultaneously enumerated for all experimental conditions after 3 to 4 wk of culture (judging by the size of colonies). Mycobacterial Ags from bacilli were prepared from Mtb H37Ra, H37Rv, and C strain in log phase cultures. Bacilli washed (three times), pelleted, and resuspended in 3 ml of UFM were irradiated by 10,000 rad of gamma irradiation, sonicated for 10 min, and heated at 65°C for 15 min. Protein content was determined by the method of Bradford (Bio-Rad, Bedford, MA). The mycobacteria Ag/lysate was stored at -70°C.
PBMC, monocyte-derived macrophages, and priming with IFN-
PBMC were separated from heparin or citrate venous blood by
Ficoll-Hypaque (9%:33%) gradient centrifugation. PBMC washed (three
times) in NS and resuspended UFM supplemented with 2% human heated AB
serum (hABS) were aliquoted as 2 x 106 PBMC onto
round-glass coverslips in 24-well plates and incubated at 37°C in 5%
CO2 for 2 h. Nonadherent cells were removed from
adherent monocytes by washing (three times) with NS containing 1% hABS
indicated and replaced with 1 ml of UFM with 2% hABS. IFN- was
added or not to the monocyte monolayers and cultured at 37°C in 5%
CO2. After 4 days, monocyte-derived macrophage (MDM) were
infected with Mtb and cocultured with autologous PBL as
detailed below.
Ag and IFN priming of PBL, and proliferative response to purified protein derivative (PPD)
PBMC (106 cells/ml) were cultured at 37°C in 5%
CO2 in UFM with 2% hABS or medium containing either 1
µg/ml of Mtb H37Ra lysate, IFN- (500 U/ml), or the
combination. After 4 days, the nonadherent cells were harvested, washed
once to remove the UFM medium, and resuspended in RPMI 1640 with 2%
BSA. PBL primed or not with either Mtb H37Ra lysate (1
µg/ml), IFN- (500 U/ml), or the combination were added to MDM
infected with Mtb as detailed below. Because the history of
prior exposure to Mtb is unknown in blood donors from the
New York Blood Center, New York, NY (NYBC), we measured T cell
proliferative response to PPD in PBMC from 12 donors in parallel to
assays in which their PBMC were used to study induction of
anti-Mtb (illustrated in Figs. 2
and 4). As a
comparison, PBMC from 5 PPD-reactive (
10 mm induration) and 5
PPD-nonreactive health care personnel (HCP) and 12 NYBC donors with
unknown PPD status were challenged in vitro with PPD (5 µg/m, 7
days). PPD-reactive HCP showed a stimulation index of 85 ± 27
(fold of PPD-induced uptake of [3H]thymidine compared
with medium). In contrast, PPD nonreactive HCP and 12 NYBC donors in
whom PPD status was unknown showed a PPD-induced stimulation index of
<2. These data suggest that the NYBC donors studied did not have
PPD-specific memory T cells, and it is likely that these donors had no
prior exposure to Mtb (K. C. Gomez et al., manuscript
in preparation).
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MDM (2 x 105 per coverslip) were washed
twice with warm NS containing 1% hABS and replaced with 500 µl of
RPMI 1640 supplemented with 2% BSA. MDM exposed to
199105 bacteria (0.5 to 1.0 CFU per MDM) for
2 h at 37°C at 5% CO2 were gently washed (three
times) with warm NS containing 1% hABS to remove Mtb.
Preliminary experiments showed that approximately 10% of the input
inoculum (tested from 0.05 to 2.0 CFU per macrophage) remained
associated with MDM (cultured for 3 to 5 days). Autologous PBL at
5 x 105 were added to each well containing
Mtb-infected macrophages pretreated or not with IFN- (500
U/ml) (designated as "cocultures"). The PBL consisted of naive PBL
or PBL primed with either IFN- (500 U/ml), Mtb lysate (1
µg/ml), or IFN- plus Mtb lysate (IFN-/Mtb
lysate). Mtb-infected macrophages or cocultures were
additionally treated or not with IFN- at indicated concentrations
and cultured at 37°C in 5% CO2 for designated times. As
a negative control, uninfected macrophages were similarly maintained.
Each experimental condition was performed in duplicate or triplicate.
In some experiments the stock IFN- (5 x 105 U/ml)
was suspended in medium containing antibiotics, 100 U/ml penicillin,
and 100 µg/ml streptomycin. In experiments in which the stock IFN-
contained antibiotics, IFN--treated macrophages had growth of
Mtb similar to infected macrophages cultured in medium,
whereas Mtb-infected macrophages cocultured with primed PBL
and further treated with IFN- had significant reduction of CFU of
Mtb (see Figs. 1 and 4
). Moreover, aminoglycosides penetrate
poorly into the phagolysosome.
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Mtb growth in 7H9 mycobacterial medium, RPMI 1640 tissue culture medium, and binding and growth in macrophages
To establish growth rates of Mtb, macrophages were
infected with Mtb (0.5 CFU/macrophage or 10 x
104 CFU/ml) while 7H9 broth and tissue culture medium were
inoculated with 1 x 104 CFU/ml. In replicate
cultures, the numbers of viable tubercle bacilli were quantitated in
these conditions at designated times, and assessed for binding. To
assess for binding of Mtb to macrophages, macrophages on
coverslips after 2 h of infection and washed (three times) were
removed, fixed for 10 min in 100% methanol, and stained with Kinyoun
Carbol Fuchsin. To enumerate the number of mycobacteria bacilli that
are macrophage associated, we counted a minimum of 100 macrophages, and
assessed the distribution of acid-fast bacilli within the macrophages
as the number of macrophages with 0, 1 to 5, 6 to 10, or 10 bacteria.
In preliminary experiments, macrophages pretreated or not with IFN-
had similar visual distribution of cell-associated mycobacteria.
Measurement of immune cell viability and profile of T cell subsets
To assess whether changes in bacillary counts may be due to killing of mycobacteria-laden macrophages, cell viability was determined in Mbt-infected macrophages or cocultures of macrophages and PBL. Using the ability of fluorescent compounds, calcein AM, and ethidium homodimer (EthD-1) to differentiate, respectively, viable or dead/dying cells, monolayers washed (one time) were treated with fluorescent dyes, 2 µM calcein AM, and 4 µM EthD-1 as instructed by the manufacturer (Molecular Probes) for 30 min. Monolayers were washed (two times) with PBS and retained dyes were quantitated by Cytofluor Reader (Millipore, Bedford, MA) using excitation wavelengths of 485 nm for calcein and 530 nm for EthD-1, and emission wavelength of 530 nm for calcein and 645 nm for EthD-1. Preliminary experiments to establish the assay used adherent monocytes and cells permeabilized with 0.2% Triton X-100.
T cell subset profiles were performed in freshly isolated PBMC, or detached cells from Mtb-infected cocultures were washed, incubated with anti-CD3 and anti-CD4 or anti-CD8 mAb, washed, fixed in 1% paraformaldehyde in PBS, and subjected to FACS analysis.
Detection of biologically active TGF-ß and IL-10, IL-12 p40, and IL-12 p70 by EIA
TGF-ß activity in cell supernatant was assayed by determining the inhibition of tritiated thymidine incorporation in CCL64 mink lung fibroblasts. An aliquot (20 µl) was diluted into 980 µl of CCL64 culture medium (medium 199 plus 10% FBS and 50 µg/ml gentamicin) and added to CCL64 cells plated 24 h previously at 1 x 104 cells per well of a 96-well plate (100 µl/well in triplicate) (21). After 18 h at 37°C, the medium was carefully decanted and 100 µl/ml tritiated thymidine in M199 medium was applied for an additional 4 h. Incorporation of tritiated thymidine into DNA was quantitated by semiautomatic techniques (22). The reduction of >60,000 cpm is maximally inhibited by as little as 5 ng/ml TGF-ß to <3000 cpm. Half-maximal inhibition is typically observed at 50 pg/ml with a detection threshold of 1 pg/ml or less of TGF-ß, and anti-TGF-ß mAb, which neutralized the inhibitory activity, served as a control. For assay of IL-10, IL-12 p40, and IL-12 p70, commercial kits were used as directed by the manufacturer, and OD was measured by EIA plate reader (Bio-Tek Instruments, Winooski, VT) at 450 nm with 590 nm as a reference.
RT-PCR for human inducible nitric oxide synthase (iNOS, NOS2)
Macrophages, Mtb-infected macrophages, or cocultures containing Mtb-infected macrophages and primed PBL were harvested and suspended in RNA Stat-60 (Tel Test-B, Friendswood, TX) at -70°C. Human 293 cells transfected with NOS2 cDNA or vector alone (gifts of Drs. Carl Nathan and Qiao-wen Xie, Cornell University Medical College) constituted positive and negative controls (23, 24, 25). The methods for detection of iNOS were as previously described (23). In brief, total RNA (1 µg) was reverse-transcribed using oligo(dT)16 and PCR performed by standard methods (GeneAmp System 9600; Perkin-Elmer, Norwalk, CT). First-round PCR was conducted with 20% of the cDNA using oligonucleotide with primers from exon 1 (5'-CAC CTT TGA TGA GGG GAC-3') and exon 4 (5'-GCA TCC AGC TTG ACC AG-3') of the human NOS2. For nested PCR, 4% of the first-round product was amplified with primers from exon 1 (5'-ATG AGG GGA CTG GGC AGT TC-3'). After electrophoresis in 1.5% agarose, reaction products (10 µl) were transferred to nylon membranes (Nytran-Plus; Schleicher & Schuell, Keene, NH) and autoradiographed after probing with 32P-labeled oligonucleotide derived from a sequence of NOS2 internal to the primers (5'-CCT TAC CCC GGG GAG GCA GTG CAG CCA G-3'). For ß-actin, a single round PCR was performed using primers from the third exon (5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3') and six intron (5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3') (Clontech, Palo Alto, CA). The ß-actin amplicons were resolved by electrophoresis in 1.5% agarose and directly stained with ethidium bromide.
Statistical analysis
Results were analyzed using either Student’s paired t test (two-tailed), Kruskall Wallis test, and ANOVA with a p value <0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first sought to determine intrinsic rates of Mtb
replication, and to evaluate whether human macrophages possess
anti-mycobacterial activity. Illustrated in Figure 1 is the kinetics of growth of
Mtb in 7H9 broth and RPMI 1640 medium, and by day 4 of
culture, bacilli growth had reached, respectively, 140-fold and
7.0-fold above the initial inoculum. Incremental growth of
Mtb in macrophages was also noted over 4 days regardless of
postinfection treatment with IFN-. As shown in Figure 1, intracellular growth of Mtb in macrophages or
IFN--treated macrophages after 4 days of culture was five- and
six-fold higher than at 2 h after infection, respectively
(p < 0.01), despite the finding that the
initial number of Mtb associated with IFN--treated
macrophages was slightly lower than untreated macrophages (2.6 ±
2.0 x 104 CFU/ml vs 5.2 ± 2.1 x
104, respectively, p > 0.05).
Induction of anti-mycobacterial activity by coculture with
primed PBL and IFN-
Control of Mtb infection in vivo is associated with
granuloma formation. Histologically, the tuberculous granuloma is
composed of a central area of macrophages within a palisade of
lymphocytes. We reason that the inability to induce in vitro
anti-mycobacterial activity in IFN--treated macrophages is due
to the requirement for activation by lymphocytes primed with Ags of
Mtb and IFN-. To reconstitute the in vivo milieu, we
assessed anti-mycobacterial activity in Mtb-infected
macrophages cocultured with autologous naïve PBL or PBL primed
with IFN-/M. tuberculosis H37Ra lysate, and we also
evaluated the additional contribution by IFN- treatment of the
infected cocultures. Cocultures of macrophages infected with
Mtb H37Ra and naïve PBL showed a 26-fold increase in
bacilli count above the bacilli count at 2 h after infection
(135 ± 14.3 x 104 CFU/ml vs 5.2 ±
2.1 x 104 CFU/ml). Cocultures of
Mtb-infected macrophages and PBL primed with IFN- (500
U/ml) showed a similar although slightly lower bacilli count (110
± 33.8 x 104 CFU/ml, p > 0.05). In
contrast, cocultures of macrophages and PBL primed with
IFN-/Mtb lysate significantly enhanced the growth of
Mtb H37Ra, which after 4 days of culture was 58-fold greater
than at 2 h of infection (301 ± 30.5 x 104
CFU/ml, p < 0.01). Cocultures of macrophages and PBL
primed with Mtb lysate showed similar growth of
Mtb (data not shown).
We next evaluated whether IFN- treatment of cocultures containing
PBL and Mtb-infected macrophages induced
anti-mycobacterial activity. (Fig. 2). Compared with cocultures without
IFN-, IFN- treatment of cocultures resulted in a significant
reduction of Mtb H37Ra. The bacilli count in IFN--treated
cocultures containing naïve PBL, PBL primed with IFN-, or
IFN-/Mtb lysate were, respectively, 25.4 ± 6.5,
16.2 ± 4.4, and 7.2 ± 1.9 x 104 CFU/ml
(p < 0.03 for each condition with IFN- vs
no IFN- (n = 8 to 26). Thus, IFN- treatment of
cocultures containing PBL primed with IFN-/Mtb lysate
reduced mycobacterial growth by 42-fold (1.8 log reduction) when
compared with cocultures not treated with IFN-. Moreover, IFN-
treatment of cocultures containing PBL primed with
IFN-/Mtb lysate restricted bacterial growth, resulting in
a bacillus count at 4 days that was similar to the count at 2 h
after infection (7.2 x 104 CFU/ml vs 5.2 x
104 CFU/ml), and significantly lower than cocultures
containing naïve PBL (25.4 ± 6.5 CFU/ml,
p < 0.05 (Student’s t test)).
Kinetics of induction of anti-mycobacterial activity by
coculturing with primed PBL and IFN-
As shown in Figure 3, a significant
time-dependent growth of Mtb was observed in macrophage
cultures or cocultures of macrophages and naïve PBL
(p < 0.05, Kruskall Wallis test). In
cocultures of macrophages and naïve PBL, we observed a 10-fold
increase in mycobacterium count (from 4.2 ± 1.3 x
104 CFU/ml at 2 h to 43.7 ± 6.4 x
104 CFU/ml at day 4 (n = 5,
p < 0.004, Kruskall Wallis test). The bacilli count at
day 4 in cocultures of macrophages and naïve PBL was slightly
higher than that in macrophages or macrophages primed with IFN-
(Fig. 3). In contrast, additional IFN- treatment of macrophages
cocultured with IFN-/Mtb lysate-primed PBL
restricted the growth of Mtb, permitted only a 2.4-fold
increase in bacilli count at 4 days (6.3 x 104 CFU/ml
vs 2.6 x 104 CFU/ml at 2 h after infection,
p > 0.05). Importantly, omission of IFN- treatment
from cocultures containing PBL primed with IFN-/Mtb
lysate resulted in a 115-fold increase in bacilli growth (compared with
2 h after infection and to IFN--treated cocultures containing
PBL primed with IFN-/Mtb lysate, p <
0.01, Student’s t test, Fig. 3).
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dose on intracellular growth of Mtb
We next examined the effect of varying doses of IFN- treatment
of cocultures of Mtb-infected macrophages and PBL on
anti-mycobacterial activity. As seen in Figure 4, a dose-dependent induction of
anti-mycobacterial activity was observed. Compared with no IFN-
treatment of the cocultures, IFN- (2000 U/ml and 1000 U/ml)-treated
cocultures of Mtb-infected macrophages showed a striking
inhibition in Mtb growth of 470-fold (2.5 log reduction,
140 x 104 CFU/ml vs 0.3 x 104
CFU/ml) and 186-fold, respectively. Treatment with higher doses of
IFN- triggered bactericidal activity. Thus, compared with uptake of
the tubercle bacillus at 2 h (2.6 ± 0.4 x
104 CFU/ml), IFN- at 1000 and 2000 U/ml reduced the
bacilli count by 4- and 10-fold, respectively, (0.7 x
104 CFU/ml and 0.2 x 104 CFU/ml,
p = 0.07 and p < 0.05, respectively,
n = 4) (Fig. 4). In contrast, at lower doses (100 and
500 U/ml) IFN- achieved bacteriostasis but significantly reduced
mycobacterial growth by 21- and 40-fold, respectively, when compared
with cocultures of Mtb-infected macrophages and
naïve PBL not additionally treated with IFN-
(p < 0.05 for all IFN- doses;
n = 6).
Anti-mycobacterial activity against M. tuberculosis C strain and H37Rv
To determine whether the in vitro cellular capacity to kill
Mtb-attenuated strain, H37Ra, can be extended to virulent
strains of Mtb, we evaluated the C strain, a clinical
isolate, and H37Rv, a virulent strain of Mtb. As shown in
Figure 5A, human macrophages
stimulated for 4 days with IFN- (500 U/ml) were unable to control
the growth of the C strain of Mtb. Bacterial recovery from
these macrophages at the end of 4 days was ninefold greater than
bacillus uptake at 2 h (87.6 x 104 CFU/ml vs
9.6 x 104 CFU/ml, respectively; p <
0.05, n = 3). The addition of IFN- (500 U/ml) to
cocultures of C strain-infected macrophages and PBL primed with
IFN-/C strain lysate significantly reduced the growth of virulent C
strain by 2.5-fold (87.6 x 104 CFU/ml vs 17.5 x
104 CFU/ml; p < 0.05, n =
4, Fig. 5A). In contrast, omission of IFN- treatment of
the cocultures of infected macrophages and PBL primed with
IFN-/Mtb C strain lysate resulted in 15-fold increase
in bacillus growth (145 x 104 CFU/ml vs 9.6 x
104 CFU/ml) after 4 days. Increasing the postinfection
treatment dose of IFN- from 500 U/ml to 1000 U/ml reduced the growth
of Mtb after 4 days to threefold below the CFU at 2
h of infection (3.2 x 104 CFU/ml, n =
2) (data not shown). Illustrated in Figure 5B, a profile of
anti-mycobacterial activity for Mtb H37Rv similar to
that of the C strain was observed. Compared with macrophages cultured
in medium, a 36-fold and a 4.5-fold growth increase of Mtb
H37Rv was noted, respectively, in cocultures of macrophages and
IFN-/Mtb lysate-primed PBL and in cocultures of
macrophages and naive (nonprimed) PBL (p <
0.03). In contrast, the addition of IFN- to cocultures of
macrophages and PBL primed with IFN-/Mtb lysate resulted
in a 4-fold and 30-fold reduction in bacillary count when compared with
cocultures of macrophages and naïve PBL, or PBL primed with
IFN-/Mtb lysate, respectively (Fig. 5B).
Similar uptake of bacilli at 2 h was noted in cocultures of
macrophages and naïve PBL or PBL primed with IFN-/
Mtb lysate.
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We assessed whether changes in bacilli count could be solely
explained by death of macrophages laden with mycobacteria. Figure 6A shows the fluorescence
emission values representing the number of live and dead cells. The
proportion of live and dead cells was similar between
Mtb-infected macrophages and Mtb-infected
macrophage cocultures containing PBL primed with IFN-/Mtb
lysate and between infected cultures not treated or additionally
treated with IFN-.
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B,
Mtb-infected macrophages cocultured with PBL primed with
Mtb lysate/IFN- had the highest percentages of CD4 and
CD8 T cells when compared with freshly isolated PBMC, or
Mtb-infected cocultures containing PBL primed with IFN-
or PBL cultured in medium. Although treatment of the infected coculture
with IFN- reduced the percentages of CD4 and CD8 T cells in all
conditions, the percentages of CD4 and CD8 T cells in infected
macrophage cocultures containing PBL primed with Mtb
lysate/IFN- remained higher than cocultures containing PBL primed
with IFN- alone. Cytokine network in Mtb-infected cocultures
The induction of TGF-ß by Mtb infection of human
macrophages has been reported, and its ability to down-modulate
macrophage effector functions is thought to play a role in the
pathogenesis of tuberculosis (26, 27, 28). We assessed TGF-ß levels in
the in vitro system. A biologic assay was used to detect active TGF-ß
because the regulation of this ligand is by activation of an inactive
precursor molecule rather than by up-regulation of ligand production.
As shown in Figure 7A,
macrophage infection by Mtb rapidly produced active
TGF-ß (n = 3). The presence of PBL primed with
Mtb lysate/IFN- reduced TGF-ß to a lower level.
Treatment of the coculture with IFN- further lowered the amount
of active TGF-ß.
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B, high levels of IL-10 and IL-12
p40 were detected in Mtb-infected macrophage cultures and in
cocultures containing infected macrophages and PBL primed with
Mtb lysate/IFN-, although exceedingly low levels of IL-12
p70 were detected in both conditions. IFN- treatment significantly
reduced IL-10, induced moderate levels of IL-12 p70, and increased the
amounts of IL-12 p40 production in cocultures containing
Mtb-infected macrophages and PBL primed with Mtb
lysate/IFN- or in Mtb-infected macrophage cultures. The
induction of IL-10 was similarly seen in macrophages infected with
either Mtb virulent strains H37Rv or C (data not shown). The
induction of IL-10 is specific to Mtb-infection because
uninfected macrophages and macrophages cultured with PPD of
Mtb produced very low amounts of IL-10.
We next correlated the level of IL-10 and IL-12 production with the
bacillary counts obtained from each of the cell coculture conditions.
Similar to earlier experiments, Mtb-infected macrophages
cocultured with PBL primed with Mtb lysate/IFN- showed
the highest bacilli count (310 ± 80 x 104
CFU/ml) and the highest IL-10, but nearly undetectable levels of IL-12
p70 (Fig. 7B). In contrast, IFN- treatment of cocultures
containing infected macrophages and PBL primed with Mtb
lysate/IFN- reduced bacilli count by 41.3-fold, and decreased IL-10
by 5.7-fold, while significantly enhancing the production of IL-12 p70
by 50-fold (n = 3, Fig. 7B). However, in
the absence of primed PBL, similar numbers of bacilli were recovered
regardless of IFN- treatment of Mtb-infected (8.7 x
104 CFU/ml and 14.5 x 104 CFU/ml,
respectively) despite the ability of IFN- to modulate IL-10 and
IL-12 levels (Figs. 1 and 7B). The data suggest that the
effect of IFN- on IL-12 p70 and IL-10 required PBL to modulate
macrophages to effectively control intracellular bacilli.
iNOS (NOS2) and control of Mtb
Expression of iNOS in mice has been shown to correlate with
limiting mycobacteria dissemination by macrophages (13, 14, 15). In
addition, expression of iNOS was recently shown in human lung
macrophages from cases with active pulmonary tuberculosis and in lung
macrophages from patients with inflammatory lung disease upon in vitro
infection with M. bovis BCG but not in peripheral blood
macrophages (23, 39, 40). We therefore examined by RT-PCR the
expression of nitric oxide synthase by cells in the in vitro system. As
shown in Figure 8, a time-dependent
induction of nitric oxide synthase was noted. A discernible amount of
nitric oxide synthase mRNA was seen in IFN--treated
Mtb-infected macrophages, while none was found in
Mtb-infected macrophages not given IFN-. The highest
amount of nitric oxide synthase mRNA was found in IFN--treated
cocultures containing Mtb-infected macrophages and PBL
primed with Mtb lysate/IFN-, while a faint amount of
nitric oxide synthase mRNA was noted in cocultures not treated further
with IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation is insufficient to
induce anti-Mtb activity of human macrophages (19, 40).
We speculated that the lack of in vitro anti-Mtb
activity of human macrophages shown by earlier investigators and by
this study was due to the absence of additional cellular and host
immune modulators. This reasoning is based on the histology finding
that the tuberculous granuloma in which Mtb has been
contained is composed of centrally located tissue macrophages harboring
tubercle bacilli surrounded by a palisade of lymphocytes and other
mononuclear cells. We therefore reconstituted such a picture by
coculturing Mtb-infected macrophages with Mtb
lysate/IFN--primed PBL and further activating these cells by
IFN-.
We report the development of an in vitro model of Mtb
infection whereby human macrophages are activated to reduce the growth
of Mtb by 2.5 logs CFU or >40-fold. Anti-Mtb
activity in this model required coculturing infected macrophages with
autologous PBL and the additional treatment of the coculture with
IFN-. Omission of Mtb Ag-primed PBL or further treatment
of the infected coculture with IFN- resulted in unrestricted growth
of Mtb (Figs. 2 and 4). The requirement of IFN- is
further supported by the finding that the restriction of bacillus
growth by IFN- is dose dependent in that the reduction of bacillus
growth by 2000 U/ml of IFN- reached 2.5 logs when compared with
similar cocultures lacking IFN- (Fig. 4). Moreover, we found that in
response to doses of 2000 and 1000 U/ml of IFN-, bactericidal
activity in cocultures containing primed PBL was achieved (10- and
4-fold reduction from initial inoculum, respectively) while lower doses
were bacteriostatic (Fig. 4).
Our experiments indicated that the highest growth of Mtb was
found in cocultures of macrophages and PBL primed with IFN- plus
Mtb lysate (Fig. 2). One potential explanation may be that
one or more Mtb Ags triggered the production of cytokines,
such as IL-10, and facilitated the development of T cells. We therefore
examined for the production of IL-10 by Mtb-infected
macrophages or by cocultures containing primed PBL. Surprisingly, high
levels of IL-10 and nearly undetectable amounts of IL-12 p70 were found
in culture supernatant from Mtb-infected macrophages and in
cocultures containing macrophages and PBL primed with Mtb
lysate/IFN- (Fig. 7B). The production of IL-10 appears to
be induced by Mtb infection because neither macrophages
cultured in medium nor medium containing PPD of Mtb produced
significant amounts of IL-10. We suspected that high levels of IL-10 in
the context of low IL-12 p70 triggered by Mtb facilitated
the expansion of a subset of T cells. This is suggested by our finding
that the highest percentage of CD4 and CD8 T cells in association with
high IL-10 levels was seen in cocultures containing
Mtb-infected macrophages and PBL primed with Mtb
lysate/IFN- (Fig. 6B).
In contrast to Mtb-infected cocultures not treated with
IFN-, additional treatment of the cocultures with IFN- reduced
IL-10 and enhanced IL-12 p70 production (Fig. 7B). This
change in cytokine profile resulting from IFN- is associated with
lowering of the percentages of CD4 and CD8 T cells in cocultures
containing Mtb-infected macrophages and Mtb
lysate/IFN--primed PBL as compared with parallel cocultures not
treated with IFN- (Fig. 6B). Reducing IL-10 and
increasing IL-12 p70 production permitted the unopposed activation by
IFN- resulting in high anti-Mtb activity. However,
activation by IFN- was not due solely to suppression of IL-10 and
induction of IL-12 p70 production, but required primed lymphocytes
because anti-Mtb activity was not seen in
IFN--activated macrophages infected with Mtb; rather,
anti-Mtb activity was seen only in cocultures containing
PBL (Figs. 1, 2, and 7B). The participation of PBL
(presumably T cells) in these interactions is also corroborated by our
finding that active TGF-ß levels were lowered in
Mtb-infected macrophage cocultures containing PBL (Fig. 7A).
It is likely that high IL-10 production in Mtb-infected
macrophage cultures or infected cocultures played a pathologic role.
This interpretation is corroborated by the recent report that IL-10
transgenic mice are susceptible to disseminated M. bovis BCG
while wild-type mice restricted the infection (31). Of note is that
spleen cells from IL-10 transgenic mice compared with cells from
wild-type mice upon activation expressed normal levels of IL-2 and
IFN- and only 4-fold higher IL-10. This amount of IL-10
overproduction was sufficient to transform a BCG resistant to a
susceptible phenotype (31). Such a case can also be made for human
tuberculosis, in that we have observed the coexpression of IL-10 with
IL-2, IFN-, and nitric oxide synthase by lung cells from patients
with active pulmonary tuberculosis (23, 41). In addition to IL-10, we
also found high amounts of IL-12 p40. Although we cannot exclude a
differential sensitivity in the ability of the assay to detect IL-12
p40 and IL-12 p70, IL-12 p40 homodimer has been shown to bind to IL-12R
and to antagonize IL-12 p70 activation (42, 43). Moreover, in mice,
IL-12 p40 homodimers were reported to block the tumoricidal activity of
IL-12 p70 (44). Thus, should the detected high IL-12 p40 exist as a
homodimer in this in vitro model, it may also have a pathologic
function. In addition, as previously reported, active TGF-ß from
Mtb-infected macrophages likely also contributed to
macrophage deactivation (26, 27, 28). However, in this model, coculture of
infected macrophages with PBL appeared to suppress active TGF-ß
levels.
In this study, high level expression of iNOS was observed in
association with anti-Mtb activity found in
IFN--treated cocultures containing Mtb-infected
macrophages and Mtb lysate/IFN--primed PBL. In the
absence of IFN- treatment of the Mtb-infected cocultures,
the low level nitric oxide synthase detected was ineffective as
indicated by the finding that these cocultures had the highest bacillus
load (Figs. 2 and 8). Although we observed that IFN--activated
Mtb-infected macrophages expressed nitric oxide synthase,
significant anti-mycobacterial activity was not demonstrated (Figs. 1 and 8). In contrast, lung macrophages from patients with lung disease
already primed by inflammatory mediators, such as in idiopathic
pulmonary fibrosis, required only the in vitro signal provided by
M. bovis BCG infection to trigger iNOS gene expression and
enzymatic activity (39). These data suggest that human iNOS gene
expression and/or its activity may be regulated by several signals,
thus accounting for the detection of iNOS activity in lung macrophages
from patients with active tuberculosis; whereas the induction of iNOS
in alveolar macrophages from inflamed lung needed only in vitro BCG
infection, and in peripheral blood macrophages iNOS expression required
Mtb infection and coculture with IFN- and primed PBL
(i.e., the in vitro model). Prior reports did not demonstrate iNOS in
macrophages (23, 39, 40). Whether the in vitro anti-Mtb
activity found by this report is solely mediated by iNOS, and whether
dual signaling induced the expression of iNOS gene with functional
activity were not examined. Similarly, what T cell subsets and how they
transmit the signal to activate macrophages were not examined. However,
the establishment of an in vitro model will facilitate our
understanding of the complex cellular and cytokine network required for
maximal anti-Mtb activity.
We believe that our present findings represent primary immune response
following infection because the New York blood donor population is
predominantly middle to upper middle class and white (Dr. Celso Bianco,
New York Blood Center, unpublished observations). In such a population,
the overall incidence of tuberculosis is 16 per 100,000 which
translates to a PPD-positive rate of less than 2% (assuming infection
to disease ratio of 1 to 100) (45). Thus, the consistent finding of
inducible anti-mycobacterial activity in all experiments in which
donors have a high likelihood of no prior exposure to Mtb
and in which 12 NYBC donors tested showed lack of in vitro recall
response to PPD (e.g., proliferation and IFN- production) strongly
suggests that our findings reflect the induction of primary immune
response. Moreover, the requirement for IFN- and high IL-10
production also argue for a primary response because if the donors’
cells contained memory cells, repriming with Ag should have expanded
these memory CD4 T cells and increased the in vitro production of
IFN-, and thus should have obviated the need for additional
treatment of the cocultures with IFN-.
Our finding that IFN- is required for induction of
anti-Mtb activity in cocultures of macrophages and
lymphocytes confirms the critical role of IFN- in defense against
Mtb. For example, administration of IFN- to mice leads to
an increase in resistance to challenge with Mtb and M.
bovis (18). In contrast, mice with homologous knockouts of IFN-
ligand or receptor genes infected with a sublethal dose of M.
bovis showed increased mortality and bacterial numbers in infected
organs (14, 15). Recently, mutation of the IFN- receptor that led to
a functionless receptor was similarly associated with disseminated
mycobacteria or BCG infection (16, 17). Moreover, IFN- treatment has
been shown to improve clinical outcome in persons with nontuberculous
mycobacterial disease (46). Furthermore, HIV-1-negative patients with
disseminated or chronic cavitary pulmonary infection caused by atypical
mycobacteria have now had IFN- added to their chemotherapeutic
regimens (47), and IFN- is being tested as an adjuvant for therapy
of patients with drug-resistant tuberculosis (S. Holland, unpublished
observations).
The availability of an in vitro model will facilitate the delineation
of mechanisms by which cytokine-modulators, in addition to IFN-,
activate anti-mycobacterial activity. We speculate that IL-12 in
this system may have an additional spectrum of activity not shared by
IFN- alone because of its ability to enhance Th1 cell, NK cell, and
CD8+ activity and development, and to increase the level of
IFN- production of these cells (34). As seen with the expression of
nitric oxide synthase, we believe that additional genes involved in
effective control of Mtb will be highly expressed in
conditions in which infected macrophage cocultures containing
IFN-/Mtb Ag-primed PBL are further treated with IFN-. The
reconstitution of effective in vitro immune response to Mtb
will enable the delineation of cell constituents, host modulators, and
cell functions/effector genes critical to restrict Mtb.
Defining the mechanisms involved using such an in vitro human system
may provide novel treatment strategies for tuberculosis. Moreover, an
in vitro system may also facilitate a systematic evaluation of
mycobacterial Ag for its ability to induce effective in vitro immunity,
and thus provide a screening method for evaluating vaccine candidates.
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Acknowledgments |
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|
Footnotes |
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2 Address correspondence and reprint requests to Dr. John L. Ho, Division of International Medicine and Infectious Diseases, Cornell University Medical College, Room A-421, 1300 York Avenue, New York, NY 10021. E-mail address:
3 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; UFM, ultrafiltered medium; iNOS, inducible nitric oxide synthase; NS, normal saline; hABS, heated AB serum; MDM, monocyte-derived macrophage; NYBC, New York Blood Center; HCP, health care personnel; EthD-1, ethidium homodimer; PPD, purified protein derivative; BCG, bacillus Calmette-Guérin.
Received for publication June 5, 1997. Accepted for publication January 8, 1998.
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