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Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021;
Tuberculosis Research Section, LHD, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; and
Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, IL-10, IL-12, and IFN-
mRNA were
apparent sooner than in lungs of mice infected with the other strains.
CDC1551-infected mice survived significantly longer. These findings
were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human
monocytes were the same, but higher levels of TNF-, IL-10, IL-6, and
IL-12 were induced in monocytes after infection with CDC1551 or by
exposure of monocytes to lipid fractions from CDC1551. CD14 expression
on the surface of the monocytes was up-regulated to a greater extent by
exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent
than other M. tuberculosis isolates in terms of growth
in vivo and in vitro, but it induces a more rapid and robust host
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3
million deaths per year. Efforts to eradicate the disease are hindered
by several factors, including the prevalence of the disease in
socioeconomic populations that have limited access to diagnosis and
treatment and the development of multidrug-resistant
Mycobacterium tuberculosis strains. Outbreaks of
drug-sensitive TB can involve infection with strains of M.
tuberculosis with widely different rates of transmission (1). In
1995, an outbreak occurred in a rural area in the United States
considered to be at low risk for TB (2). The strain, designated CDC1551
or CSU 93 by the Centers for Disease Control and Prevention (CDC)
attracted special attention because of an unusually high rate of
transmission as evaluated by skin test conversion. In addition,
infected subjects had a very large skin test response to purified
protein derivative of tuberculin (PPD). Infection with CDC1551 was not
associated with an obvious increase in active TB cases, nor did any
patients have extrapulmonary disease. However, when the growth of
CDC1551 was evaluated in lungs of mice at 20 days after aerosol
infection, 100-fold higher numbers of bacilli were isolated compared
with numbers of bacilli isolated from the lungs of mice infected with
the M. tuberculosis laboratory strain Erdman (2). This led
investigators to conclude that CDC1551 had "increased virulence,"
and this clinical isolate was selected for sequencing by the National
Institutes of Health (3). In the present study, we have used in vivo and in vitro models of infection to investigate whether CDC1551 is indeed more virulent than other M. tuberculosis strains. Using an aerosol infection model, we compared the growth rates of two recent clinical isolates and two laboratory strains of M. tuberculosis in the lungs, spleen, and liver of infected mice. Simultaneously, we monitored the granulomatous response in the infected lungs, the relative cytokine mRNA levels expressed in the infected lungs, and the clinical response to infection. To confirm that the host response observed in the mouse is analogous to that of humans, we examined mycobacterial growth and the cell surface markers and cytokine response following infection of human monocytes in vitro with M. tuberculosis CDC1551 and H37Rv.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The experiments involved laboratory strain H37Rv (Trudeau Institute, Saranac Lake, NY); strain CDC1551 (Dr. T. M. Shinnick, CDC, Atlanta, GA); strain Erdman (provided as multiple vials of stock by Dr. J. Belisle, Colorado State University, Fort Collins, CO); clinical isolates HN60 and HN878 recovered from patients in Houston, TX. H37Rv and the three clinical isolates were grown for 7 days in Middlebrook 7H9 medium (Difco, Detroit, MI) containing 0.05% Tween 80 (Sigma, St. Louis, MO) at 37°C with daily agitation. All stocks at 107–108 bacilli/ml were stored frozen at -70°C until use. All procedures were performed in a laminar flow hood in a biosafety level III laboratory.
Preparation of mycobacterial fractions
Proteins. Cultures of H37Rv and CDC1551 (100 ml) grown for 7 days from a starting OD of 0.1 at 650 nm in GAS media (glucose-alanine salts at pH 6.6 contains 0.3 g/L Difco Bacto Casitone, 0.05 g/L ammonium iron(III) citrate, 4.0 g/L K2HPO4, 2.0 g/L citric acid, 1.0 g/L L-alanine, 1.2 g/L MgCl · 6H2O, 0.6 g/L K2SO4, 2.0 g/L NH4Cl, 1.80 ml of 10 M NaOH, 10.0 ml of glycerol). The filtrate was concentrated by ultrafiltration through a 10,000-m.w. cutoff filter (Millipore, Bedford, MA) to a final volume of 2.0 ml (secreted proteins). The harvested cells were then suspended in 2 ml of PBS (pH 7.5) and combined with 0.5 g of 0.1-mm-diameter glass beads and homogenized in a minibead beater apparatus (BioSpec Products, Bartlesville, OK) for 3 min. The 10,000 x g supernatant was collected (cytoplasmic proteins). The remaining cell debris was suspended in 2 ml of 0.1% Triton X-100 (Sigma), mixed for 30 min at room temperature, and then centrifuged for 2 min (cell wall-associated proteins).
Lipids. Cultures of H37Rv and CDC1551 (100 ml) were grown in Middlebrook 7H9 media to an OD (650 nm) of 0.5 (8.7 x 1013 CFU), recovered by centrifugation at 12,000 x g for 20 min, suspended in 20 ml of methanol, 0.3% aqueous sodium chloride (100:10), and extracted with 10 ml of petroleum ether for 15 min. The petroleum ether layer was removed, transferred to a glass vial, and dried under nitrogen. Two milliliters of PBS were added to the dried residue, and the vial was sonicated in a bath sonicator for a total of 5 min in 1-min bursts (apolar lipids). The remaining cells were extracted twice with 17.3 ml of chloroform, methanol, 0.3% aqueous sodium chloride (90:100:30). Centrifugation was followed by drying of the organic layer under vacuum. The residue was resuspended in 2 ml of PBS and sonicated as above (polar lipids).
Reagents
OADC supplement (sodium chloride, bovine albumin fraction V, dextrose, catalase, oleic acid) was from Becton Dickinson (Cockeysville, MD); human serum (pooled AB+) was from Gemini Bio Products (Calabasas, CA).
In vivo studies
Mice. Female 8-wk-old (C57BL/6 x DBA/2)F1 (B6D2F1) mice, free of common viral pathogens were from Charles River Laboratories (Wilmington, MA).
Aerosol Infection.
For each experiment, a vial of stock bacilli was sonicated in a water
bath sonicator (Laboratory Supplies, model G112SPIT) for 30 s to
disperse clumps and diluted in saline containing 0.04% Tween 80 to the
final concentration of 8 x 106/ml in 10 ml. Mice
were exposed to the aerosol for 30 min using a Lovelace nebulizer
(In-Tox Products, Albuquerque, NM). This implants 200–400 organisms
into the lungs of each mouse as confirmed by plating lung homogenates
3 h after infection (4). The protocol was approved by the Animal
Use and Care Committee of The Rockefeller University.
CFU assays. The growth of M. tuberculosis in the lungs, spleen, and liver of infected mice was evaluated by homogenizing the right lung, the whole liver, and spleen in saline plus 0.04% Tween 80 and plating 10-fold serial dilutions of the homogenate on Middlebrook 7H11 agar (Difco).
Histology and morphometry. The lingula and the upper lobe of the left lung of each infected mouse were fixed in 10% formalin, embedded in paraffin, and processed. Sections were stained with hematoxylin-eosin and Ziehl-Nielsen for light microscopy. Morphometry was performed with Microcomp, a computer-based image analysis system (Southern Micro Instruments, Atlanta, GA). A calibration micrometer slide was used to determine the area evaluated (square micrometers).
RT-PCR for cytokine mRNA detection in lung homogenates. The lower lobe of the left lung of each infected mouse was removed and immediately frozen. Tissues were homogenized in 3 ml of RNAzolB (Cinna/Biotecx, Houston, TX) with a tissue Polytron homogenizer. RNA was extracted according to the manufacturer’s instructions. The RT-PCR was conducted as described (5). Densitometry of the amplification bands was conducted using a Phosphorimager (Molecular Dynamics). Results were normalized to the densitometry of ß-actin and expressed as relative units or fold increase over baseline (uninfected control mouse lung).
In vitro studies
Human monocytes. PBMCs were isolated from fresh human blood (buffy coat) (New York Blood Center, New York, NY) and from healthy PPD-positive donors by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) as described previously (6). The cells were resuspended in RPMI 1640 (Life Technologies, Gaithersburg, MD), supplemented with 1% AB human serum (R1) (Gemini Bio Products, Calabasas, CA) and adjusted to 6 x 106/ml; 0.5 ml were plated in 24-well Falcon tissue culture dishes (Becton Dickinson Labware, Lincoln Park, NJ). Adherent cells were cultured in RPMI 1640 with 20% human serum (R20) at 37°C and 5% CO2 (7).
Infection of human monocytes. Adherent monocytes were infected with M. tuberculosis H37Rv and CDC1551 on day 0. Before infection, aliquots of bacteria were probe-sonicated for 20 s at low output power (model 60 sonic dismembrator, Fisher Scientific, Springfield, NJ) to disperse clumped bacilli. All the procedures involving probe sonication of M. tuberculosis were conducted within a Biological Safety Cabinet Class II located in a Biosafety Level III Laboratory. All procedures used were approved by the Laboratory Safety Committee at The Rockefeller University. The bacterial suspension, diluted in R20, was added at a multiplicity of infection of 1 viable bacillus per cell to monolayers of 3 x 105 monocytes in R20 medium and cultured for 4 days (8). At the designated time points, PBS containing 0.016% digitonin (Sigma) and 0.25% Tween 80 (Sigma) was added to each well to release mycobacteria from the cells. After 10 min at 37°C, the cultures were probe sonicated, and mycobacteria were plated on Middlebrook 7H10 agar supplemented with OADC, as described (9).
Cytokine determination. Culture supernatants from infected monocytes or from monocytes incubated with vortexed 1:50 dilutions of polar or apolar lipids obtained from M. tuberculosis H37Rv and CDC1551 were harvested, frozen at -70°C, and then assayed with commercial ELISA kits (Endogen, Boston, MA) according to the manufacturer’s instructions. Spontaneous release of cytokines was monitored by culturing monocytes in the absence of experimental infection or stimulation.
mAbs.
The following mAbs were used for flow cytometry: FITC-anti-CD3,
PE-anti-CD14, PE-anti-CD80 (B7.1), PE-anti-HLA-DR, and
PE-anti-1 (isotype) control (Becton Dickinson, San
Jose CA); FITC-anti-CD40 and PE-anti-CD86 (B7.2) (PharMingen,
San Diego, CA).
Flow cytometric analysis. Monocytes stimulated with polar lipids (see above) were incubated for 30 min on ice and then treated for 30 min with cold PBS containing 0.2% EDTA (pH 7.2). Detached cells were washed three times with cold RPMI, and the pellet was resuspended in 700 µl of R1. Approximately 1.3 x 105 cells were incubated with 10 µl of conjugated mAbs in the dark at 4°C for 15 min; washed once in cold PBS containing 1% FCS, 1% human serum and 0.01% sodium azide; fixed overnight with 1% paraformaldehyde; and protected from light at 4°C until flow cytometry (FACScan, Becton Dickinson).
Statistical analysis. The in vitro cytokine data were analyzed by a paired t test. Kaplan-Meier analysis was used to determine statistical significance of the differences in survival of mice; 95% confidence indices and the log rank test were used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Growth of M. tuberculosis strains in the lungs of mice infected by aerosol
The course of lung infection following aerosol exposure of mice to
either M. tuberculosis H37Rv, Erdman, or CDC1551 was
followed for 60 days (Fig. 1
A)
and to M. tuberculosis HN60 and HN878 for 28 days (Fig. 1B). All strains except for the Erdman strain grew
immediately in the lungs. By day 14 postinfection, H37Rv had multiplied
from log10 2.2 ± 0.1 to log10 5.8 ±
0.5, CDC1551 from log10 2.4 ± 0.4 to
log10 6.6 ± 0.4, HN60 from log10 2.9
± 0.3 to log10 6.8 ± 0.3, and HN878 from
log10 3.0 ± 0.3 to log10 7.1 ± 0.2.
In contrast, the Erdman strain showed a delay in initial growth and
then multiplied from log10 2.4 ± 0.2 to
log10 4.1 ± 0.4 by day 14 (Fig. 1A). In
these experiments, the estimated doubling times during the first 14
days of infection were similar for CDC1551, HN60, HN878, and H37Rv, but
the doubling time was twice as long for Erdman (Table I). From 14 to 21 days, the CDC1551
strain grew more slowly than the other strains, suggesting that CDC1551
was subject to earlier growth restriction by the immune response of the
host. This is supported by the change in doubling time from 25 h
(0–14 days) to 105 h (14–21 days). The other M.
tuberculosis strains continued growing at about the same initial
rates up to day 21 postinfection (Table I). CDC1551, H37Rv, HN60, and
HN878 CFU in the lungs decreased slightly from day 21 while Erdman
continued growing albeit very slowly (see Fig. 1, A and
B, and Table I).
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Granulomatous response in the lungs
Granuloma formation in the lungs infected with each of the strains
was then examined. Small, well-organized granulomas were observed in
histological sections of the lungs at day 14 only in CDC1551-infected
mice (Fig. 1
C). The calculated volume of these granulomas
was 0.03 mm3. In contrast, H37Rv-, HN60-, HN878-, and
Erdman-infected mice did not have organized granulomas. Rather, the
lungs of these animals had small cell aggregates of about 0.01, 0.008,
0.001, and 0.003 mm3, respectively. At 14 days
postinfection, the total number of granulomas, cell aggregates detected
in all histological sections of lungs was 2-fold higher for the
CDC1551-, HN60-, and HN878-infected mice than for the mice infected
with H37Rv (13.3 ± 6.4 for CDC1551, 16.6 ± 8 for HN60,
15.3 ± 9 for HN878, and 6.3 ± 2 for H37Rv). By 21 and 28
days, the size and numbers of granulomas in the lungs of mice infected
with H37Rv, HN60, and HN878 were comparable with those seen in the
lungs of CDC1551-infected mice (Fig. 1, C and D).
Well-formed, differentiated granulomas containing heavily infected
macrophages and cuffs of lymphocytes were observed in the lungs by 28
days. In contrast, in the lungs of mice infected with Erdman,
granulomas developed much more slowly and did not achieve the size
observed in the lungs of mice infected with the other strains (60-day
experiment) (Fig. 1, C and D).
Survival of mice
To determine whether there were differences in survival, mice were
infected via aerosol with CDC1551, HN60, HN878, or H37Rv and monitored
for >250 days. Mice infected with HN60 and HN878 began to die at about
day 30, whereas mice infected with H37Rv did not begin to die until
about day 80 (Fig. 2). The mean survival
times were 177 days for mice infected with HN60 and 126 days for HN878
(95% confidence intervals 151–202 days and 99–153 days,
respectively). The mean survival time for mice infected with H37Rv was
185 days (95% confidence interval 164–205 days). Survival of mice
infected with HN878 was significantly shorter than survival of mice
infected with HN60 (p = 0.001). In contrast,
mice infected with CDC1551 survived significantly longer than mice
infected with the other strains, with a mean survival time of >250
days (median of 257, 95% confidence interval 239–275 days)
(p < 0.001 compared with all strains).
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To further evaluate the host immune response in the lungs,
cytokine production was measured with a semiquantitative assay of
specific mRNA levels (RT-PCR). We compared cytokine mRNA expression in
lungs of mice infected with each of the five strains. To visualize the
kinetics of changes in the cytokine-specific mRNA levels over the
experimental period, results were expressed as fold increase over
uninfected controls (baseline) assayed at the same time. Fig. 3 shows that there is earlier expression
(day 7) of cytokine mRNA for TNF-, IL-6, and IL-12 in
CDC1551-infected mice. This pattern of early cytokine expression was
not seen in response to infection with the other strains. By day 14,
expression of all cytokines had increased in the CDC1551-infected mice.
Thereafter, there was a transient reduction in cytokine expression (day
21–28) followed by a return to higher levels by day 60, with
especially high levels of IL-10 and IFN-. In contrast, cytokine
levels increased more slowly following infection with H37Rv, HN60,
HN878, or Erdman (Fig. 3). Fig. 4 shows a
direct comparison of the actual cytokine levels, assayed simultaneously
at 14 and 60 days after infection with CDC1551, H37Rv, and Erdman. At
day 14 postinfection, only CDC1551 elicited considerable cytokine
levels in the lungs. TNF-, IL-6, IL-10, IL-12, and IFN- were all
expressed at higher levels than in the lungs of mice infected with
H37Rv and Erdman. By 60 days postinfection, TNF- levels were similar
for the three strains. The other cytokines had increased in the lungs
of H37Rv- and Erdman-infected mice, although the extent of increase
following infection with Erdman was lower (Fig. 4).
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We then examined the response of human monocytes to infection with selected strains of M. tuberculosis. H37Rv was selected for these studies because it is the strain recently sequenced and therefore of interest for detailed biochemical and genetic analyses (11). The completion of the sequence of CDC1551 will enable direct comparison of these two strains (3).
Intracellular mycobacterial growth rates.
To compare the growth of CDC1551 with that of the laboratory strain
H37Rv, human monocytes were infected with each strain at a multiplicity
of infection of one viable bacillus per cell. In fresh human monocytes,
both M. tuberculosis strains grew at a similar rate (Fig. 5A). The calculated doubling
time of H37Rv during the 4 days of culture was 31 ± 3.8 h.
CDC1551 grew within monocytes with a doubling time of 24 ±
3.9 h.
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was detected at 24 h following infection. M.
tuberculosis CDC1551 elicited higher TNF- levels than H37Rv at
all time points (p 
0.05) (Fig. 5B). TNF- was not detectable in the culture supernatant
in the absence of experimental infection. IL-10 and IL-12 levels were
also determined in the same supernatants. Unlike TNF-, the peak of
IL-10 production was observed at 96 h after infection with either
strain (Fig. 5C). IL-10 concentrations induced by CDC1551
were higher than those induced by H37Rv. A small increase in
spontaneous release of IL-10 into culture supernatant was detected
96 h after infection. In preliminary studies, IL-12 levels were
maximal at 24 h postinfection of the monocytes. Higher levels of
this cytokine were induced by infection of the monocytes with CDC1551
compared with H37Rv (Fig. 5D). IL-12 was not detectable in
the culture supernatant of uninfected cells. Thus, in spite of similar
numbers of intracellular organisms, monocytes infected with M.
tuberculosis CDC1551 produced higher amounts of cytokines.
Therefore, the stronger cytokine response following infection of human
monocytes with CDC1551, compared with H37Rv, was analogous to the
results obtained in vivo in mice, where CDC1551 induced higher levels
of cytokine mRNA in the infected lungs. Cytokine induction and CD14 up-regulation by polar and apolar lipid fractions of M. tuberculosis CDC1551 and H37Rv in human monocytes. Previous studies have shown that mycobacterial proteins and lipids play an important role in inducing the host response (13, 14). Since the cytokine responses to infection with these two strains were different, we examined whether the two M. tuberculosis strains contained components with differing capacities to induce monocyte cytokines.
Mycobacterial fractions (polar and apolar lipids, secreted proteins,
cell wall-associated components, and lysate components) were tested for
their ability to induce monocyte cytokine production in vitro. The
relative levels of monocyte TNF- and IL-12 induction by the
different fractions of CDC1551 are shown in Table II. The lipid fractions were found to
induce 70–75% of the total activity. Therefore, the two lipid
fractions (polar and apolar) prepared from the same number of either
M. tuberculosis H37Rv or CDC1551 were compared for induction
of TNF-, IL-10, IL-12, and IL-6.
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shows the production of TNF-
and IL-12. For all cytokines tested, the response to the polar lipid
fraction of CDC1551 was higher (p 0.01) than
the response to the polar lipid fraction of H37Rv. The response to the
CDC1551 apolar lipid fraction was higher for TNF-
(p = 0.01) (Fig. 6) and IL-6
(p 0.01) (not shown). The differences in
levels of IL-10 and IL-12 induced by apolar lipids of either H37Rv or
CDC1551 were consistent but were not statistically significant
(p = 0.09). Thus, the differences in cytokine
production observed following infection with CDC1551 and H37Rv may be
due to differences in the lipid components of these M.
tuberculosis strains.
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). The lipid fraction
from CDC1551 induced higher levels of CD14 expression than did lipids
of H37Rv. CD3, CD80, CD86, HLA-DR, and CD40 levels were unaffected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies confirmed that the CDC1551 strain grew faster in mice than
the Erdman strain did. However, our results showed that two additional
recent clinical isolates and the commonly used laboratory strain,
H37Rv, grew similarly in mice with a generation time of 25–28 h
(measured from 0 to 14 days after infection) compared with the 25-h
generation time observed for CDC1551 (Fig. 1, Table I). Therefore, the
growth rate of CDC1551 is not unusually fast. In vitro in human
monocytes, H37Rv and CDC1551 showed a 20% difference in the doubling
times (31 and 24 h, respectively) (Fig. 5A). Thus, if
the growth rate of mycobacteria is considered to be an indicator of
virulence (15), then CDC1551 is not more virulent than other recent
clinical isolates or than the H37Rv laboratory strain of M.
tuberculosis. It does, however, appear to be more virulent than
the Erdman strain.
The results reported here suggest that if CDC1551 differs significantly
from other strains of M. tuberculosis, the difference is in
the ability of this strain to induce a host immune response. In the
mouse, CDC1551 induces granulomatous differentiation in the lungs at an
earlier time point than that for the other clinical isolates and H37Rv
and Erdman (Fig. 1B). In spite of what appear to be
relatively small differences in the volume of the cellular aggregates
induced in the lungs of infected mice (3–30-fold), cytokine mRNA
levels expressed in the lungs are higher and appear sooner in response
to infection with CDC1551 (Figs. 3 and 4). Consequently, this prompt
host response is associated with earlier control of CDC1551 growth in
the lungs, as shown by the rapid (14 days) establishment of chronic
stable infection with no increase in CFU in the lungs. Although these
differences are apparent for only a few weeks, they are critical for
the long term outcome, since CDC1551-infected mice survive
significantly longer than mice infected with H37Rv, HN60, and HN878
(Fig. 3) or Erdman (not shown). In addition, the results obtained in
vitro show that the differences in the host response observed in mice
are also observed in human monocytes, thereby confirming that CDC1551
induces an accelerated and more robust cytokine response.
The mechanism underlying the better protective immune response to
CDC1551 infection and improved outcome is suggested by the results of
our experiments. IFN- is an important mediator of macrophage
activation, and the regulation of this cytokine has been considered to
be central to the protective immune response (16, 17, 18). IL-12 induces
IFN- production by lymphoid cells (19, 20). The early expression of
IL-12 during T cell differentiation has been shown to favor the
development of a Th1-type protective cytokine response over a Th2-type
cytokine response (21, 22, 23). In our studies, IL-12 was expressed in the
lungs of mice infected with CDC1551 by day 7 postinfection, long before
it appeared in the lungs of mice infected with the other M.
tuberculosis strains. It appears, therefore, that the early
induction of IL-12 and the relatively high levels of IFN- expressed
in the lungs of these mice throughout the first 60 days of infection
may be responsible for the prompt local control of mycobacterial growth
and the longer survival of mice infected with CDC1551. In our monocyte
infection studies conducted in vitro, CDC1551 induced substantially
higher IL-12 production at 24 h than the level induced by H37Rv.
This in vitro observation confirms our in vivo observations. That IL-12
is actually involved in the human protective immune response to
mycobacteria is indicated by studies in humans with a genetic absence
of an intact IL-12 signaling pathway. In these individuals, monocyte
costimulation for IFN- production is defective, resulting in
disseminated Mycobacterium avium infections (24).
In addition, TNF- has also been shown to be required for the
generation of the protective host response as well as for the
generation and maintenance of granulomas (25, 26). In our studies,
earlier expression of TNF- in the lungs of CDC1551-infected mice was
associated with earlier appearance of organized granulomas. Since the
granulomatous response plays a critical role in control of TB, early
production and higher levels of TNF- in the lungs may also
contribute to the better survival of the CDC1551-infected mice.
Our observation that CDC1551 infection of mice and human monocytes
induces an earlier and more robust host response may partially explain
the observation that individuals exposed even casually to CDC1551
showed a converted skin test response with an unusually large
induration. What regulates the size of the PPD response is not known
(27, 28). The intradermal injection of PPD to previously sensitized
individuals induces the expression of a series of physiological
changes, including the emigration to and activation of lymphocytes and
monocytes at the dermal site (29). In some individuals, the tuberculin
test may result in a large, blistering, necrotic lesion. Ag-specific T
cell activation has been shown to be necessary for PPD skin test
conversion to occur. In addition, monocyte cytokine production,
particularly TNF-, has been implicated as a determinant of size and
tissue damage of the PPD response (30). We suggest that even small
numbers of infecting CDC1551 bacilli induce a cytokine response large
enough to stimulate granuloma formation and T cell migration and
activation at the site of infection. This is reflected by the high
frequency of skin test conversion. If these individuals develop active
disease, we would predict well-differentiated granulomas in their
lungs. In addition, there would be up-regulation of CD14 on the
monocytes of exposed individuals, "priming" these to respond more
vigorously to future exposure to M. tuberculosis lipid
products. These cells may release higher TNF- levels, resulting in
larger PPD responses upon skin testing. Increased TNF- responses
following BCG sensitization have been reported in mice and in humans
(31, 32).
The differential cytokine-inducing capacity of CDC1551 appears to be a
property of that strain. When crude lipid fractions of the bacilli were
tested in monocytes, they induced significantly higher TNF- and
IL-12 production and higher CD14 expression than similar fractions
prepared from H37Rv. This finding suggests that polar and/or apolar
lipids of M. tuberculosis CDC1551 may be responsible for the
differential response. At present, we do not know whether the lipid
fractions of CDC1551 are qualitatively or quantitatively different from
those of H37Rv. It is also not clear which molecule(s) may be
responsible for the differences. Studies have shown that
lipoarabinomannan, a major cell wall component of M.
tuberculosis, induces monocyte cytokine production possibly via a
CD14-dependent pathway (13, 33). However, other lipids may signal
monocyte cytokine production through a CD14-dependent pathway (34).
Future studies will be directed toward fractionating the lipids,
characterizing the active components, and identifying the specific
molecules which mediate the effects, including CD14 up-regulation,
reported here. In addition, with the completion of the sequence of
CDC1551, a direct genetic comparison of this strain with H37Rv (3) will
be possible. This information should help explain the special
properties of CDC1551.
In summary, the studies presented here provide experimental evidence indicating that M. tuberculosis CDC1551 is not unusually virulent but rather more immunogenic and that it induces a rapid and vigorous cytokine response which may account for the very high frequency and large size of PPD responses following exposure to patients infected with this M. tuberculosis isolate.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gilla Kaplan, Laboratory of Cellular Physiology and Immunology, Rockefeller University, 1230 York Avenue, New York, NY 10021. E-mail address:
3 Abbreviations used in this paper: TB, tuberculosis; CDC, Centers for Disease Control and Prevention; PPD, purified protein derivative of tuberculin.
Received for publication January 25, 1999. Accepted for publication March 17, 1999.
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References |
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) and H37Rv
(
) and two experiments for Erdman (
), HN878 (
), and HN60 (
)
(3–4 mice/time point). A t test was used for
statistical analysis (p < 0.01, granuloma size
induced by CDC1551 vs H37Rv and Erdman).
), 2.2 log10 H37Rv (•), 2.7 log10 HN878
(x), and 2.6 log10 HN60 (
), H37Rv (
). RNA extracted from uninfected lungs was used as controls (
).
Results are expressed as mean ± SD of relative density units
obtained from one representative experiment of three (three to four
mice per time point per strain) calculated as described in
Materials and Methods. To assure equivalent PCR
conditions, the assays for the mRNA obtained from the lungs of the mice
infected with the three M. tuberculosis strains
were conducted simultaneously.
) and H37Rv (