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Institut National de la Santé et de la Recherche Médicale, Unité 82, Institut National de la Santé et de la Recherche Médicale, Hôpital Bichat-Claude Bernard, Paris, France; and
Unité de Génétique Mycobactérienne, Institut Pasteur, Paris, France
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or its receptor, TNF-
or TNF receptors, IL-12, and IL-18 have all been shown to seriously
impair host immunity in murine models (5, 6, 7, 8, 9, 10, 11), and the
identification of mutations in the genes coding for IFN- and IL-12
receptors in individuals with increased sensitivity to mycobacterial
infection have confirmed the absolute requirement for these cytokines
in the control of mycobacterial infection in humans
(12, 13, 14, 15).
The mechanisms through which T cells increase the mycobactericidal
activity of macrophages remain controversial. Studies evaluating murine
macrophages in vitro have shown that cytokines produced by T cells, in
particular the combination of TNF- and IFN-, can directly
stimulate macrophage bactericidal activity, at least in part through
the induction of NO synthetase activity (16, 17, 18). Numerous
attempts to demonstrate similar effects of these cytokines on human
macrophages have failed. Whether used individually or in combination,
TNF-, IFN-, and IL-12 have not been found to reproducibly improve
the mycobactericidal activity of human macrophages
(19, 20, 21, 22). Taken together, these findings suggest that
exposure to T cell-derived cytokines alone is not sufficient for the
development of potent mycobactericidal activity in human macrophages.
Several recent studies have suggested that cell-cell interactions
between T cells and macrophages may also contribute to the development
of mycobactericidal activity (22, 23, 24).
In addition, lymphocytes may act indirectly, by fostering the formation
of well-developed immune granulomas, which are a constant feature of
effective antimycobacterial responses in both experimental models and
humans. Granulomas are composed of a central core of monocyte-derived
cells expressing characteristic morphologic features (epithelioid
macrophages and giant cells) that is surrounded by and infiltrated with
activated CD4+ and CD8+ T
cells. Cytokine secretion by T cells is required for granuloma
formation in vivo, because depletion of T cells or inactivation of
signaling by IFN-, TNF-, and IL-12 have all been shown to impair
and/or delay granuloma formation (3, 4, 5, 6, 7, 8, 9, 10, 25, 26), and the
extent of these abnormalities correlates with the overall impairment in
mycobactericidal activity observed.
The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, we have evaluated the effect of macrophage density and maturation on the intracellular growth of Mycobacterium bovis bacillus Calmette-Guérin (BCG),3 and demonstrate that culture at high density considerably improves the ability of human macrophages to control mycobacterial proliferation. The evaluation of the mechanisms involved suggests that differences in their differentiated state, not the production of autocrine/paracrine mediators, are chiefly responsible for the improved bacteriostatic activity of these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from leukapheresis concentrates obtained from healthy volunteers by centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway). Monocytes were then purified by counterflow centrifugal elutriation (27), using a J2-21 ME centrifuge and a JE-6B rotor (Beckman Instruments, Palo Alto, CA). Monocytes had a viability of >95% and a purity of at least 92% in all experiments. For some experiments, monocytes that had been frozen at -80°C in 10% DMSO were used.
To initiate experiments, monocytes were resuspended in complete medium (IMDM (Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 200 U/ml penicillin G, 1 µg/ml kanamycin (Sigma), and 20% human AB serum (Institut Jacques Boy, Reims, France)). Cells were cultured in 96-well flat-bottom plates with opaque sides and transparent bottoms (EG&G Wallac, Turku, Finland) at concentrations ranging from 1 x 104 to 2 x 105 cells/well in a final volume of 200 µl medium. In most experiments, cells were maintained at 37°C in 95% air, 5% CO2 for 3 days before infection. In some experiments, cells were cultured for 10 days before infection, in which case medium was replaced after 7 days of culture.
Infection of monocytes
The construction of the mycobacterial reporter strain used in
these studies has previously been described (19). Briefly,
to obtain this strain, M. bovis BCG 1173 P2 (Pasteur
Institute, Paris, France) was transformed with the plasmid pPV12, which
contains the mycobacterial origin of replication from
pAL5000, a kanamycin resistance gene, and the luciferase
gene Luc1 from Photinus pyralis under control of
the mycobacterial promoter Pan. Mycobacteria were grown at 37°C with
gentle shaking in 7H9 liquid medium supplemented with Middlebrook ADC
enrichment mixture (Difco, Detroit, MI) and 20 µg/ml kanamycin to an
OD600 of 
1, resuspended by pipeting, diluted
with an equal volume of 40% glycerol, and stored as 1.2-ml aliquots at
-80°C. On the day of infection, stocks were thawed, 1.2 ml complete
medium without serum were added, and aggregates were dispersed by three
cycles of sonication (50 W, 30 s, 0°C) using a Vibracell
sonicator (Sonics and Materials, Danbury, CT) followed by vortexing (5
s). Mycobacteria were pelleted by centrifugation (1800 x
g for 10 min), resuspended in 3 ml complete medium without
serum, and centrifuged (40 x g for 5 min) to pellet
residual aggregates. The top 2.5 ml of the suspension were removed, and
mycobacteria were quantified using a disposable counting chamber (Kova
Slide 10; Hycor Biomedical, Irvine, CA). In most experiments,
mycobacteria were diluted to 2 x 103
mycobacteria/µl. In experiments evaluating the effect of changes in
the multiplicity of infection, suspensions containing a 4-fold higher
and 4-fold lower concentration of organisms were also used. Most
mycobacteria in the suspension were individual organisms, although a
small proportion of aggregates containing fewer than five mycobacteria
were also present. Essentially 100% of the organisms were viable as
assessed using live/dead BacLight viability stain (Molecular Probes,
Eugene, OR).
To infect cells, culture medium was removed, and 25 µl of the mycobacterial suspension were added (e.g., 5 x 104 mycobacteria/well), followed by fresh complete medium with or without other additions as indicated below to a final volume of 200 µl. At different times after infection, 175 µl medium were removed from each well, and the plates were either frozen at -20°C (for measurement of luciferase activity) or used immediately for the evaluation of CFU. In some experiments, wells were washed three times with 200 µl warmed complete medium before evaluating luciferase activity.
Evaluation of mycobacterial growth
Determination of luciferase activity. Mycobacterial number was assessed by measuring luciferase activity produced by the mycobacterial reporter strain as previously described (19). Briefly, 96-well plates where thawed, and 75 µl lysis reagent (Promega, Madison, WI) were added to lyse the macrophages and permeabilize the mycobacteria. Luminescence was then measured during a 50-s interval using a EG&G Berthold MicroLumat LB-96P luminometer, 1.6 s after automatic injection of 100 µl of the substrate solution containing luciferin and ATP (Luciferase Assay Reagent; Promega). In all experiments, results for each experimental condition, expressed as relative light units (RLU (3)/well, are the means of triplicate determinations.
CFU. After removal of 175 µl medium from each well, 50 µl 7H9 Middlebrook medium containing 0.1% SDS and 20 µg/ml kanamycin (37°C) were added. After a 30-min incubation at 37°C, 75 µl PBS containing 20% BSA was added, and the contents of each well were resuspended by vigorous pipeting. Three serial 10-fold dilutions were prepared using 7H9 Middlebrook medium, and 75-µl aliquots were plated in triplicate on petri dishes containing 7H10 medium supplemented with Middlebrook OADC enrichment mixture (Difco) and 20 µg/ml kanamycin. Plates were incubated at 37°C for 3 wk before colonies were counted. Results are expressed as CFU/well, and in all experiments are the average of triplicate values for each experimental condition.
When mycobacterial number was evaluated in parallel 24 h after
infection based on CFU and RLU, a CFU:RLU ratio of 9.2 ± 3.7
(n = 8) was observed; this ratio was not different for
cultures containing 5 x 104 and 2 x
105 cells/well. As indicated above, cultures were
infected with 
5 x 104 mycobacteria/well.
As expected, luciferase activity 24 h after infection was 5000
RLU/well in all experiments, and the activity was similar in cultures
containing 5 x 104 and 2 x
105 cells/well.
Evaluation of morphology and cell number
Cytologic evaluation was performed on cells grown under conditions identical with those used to evaluate mycobacterial growth. To evaluate cell morphology and viability, culture medium was removed and replaced with solution containing 50 ng/ml propidium iodide and 3 µg/ml acridine orange in PBS, and cultures were examined under epifluorescent illumination using an inverted Zeiss fluorescent microscope. To identify mycobacteria, culture medium was removed, and cells were fixed with 10% formalin for 30 min, washed with 100 µl PBS, and stained for 20 min with 30 µl 5 µg/ml acridine orange and 0.7 µg/ml propidium iodide in PBS. After washing with PBS, the bottom of each well was removed using a hollow steel punch, and the disc was attached to a microscope slide using Super Glue-3. Slides were immersed in methanol, dried, restained with 25 µl of the fluorescent dye solution, and rinsed successively with PBS and methanol. The slides were examined under epifluorescent illumination using a Leitz DMRD microscope camera system (Leica, Wetzlar, Germany). DNA content of cultures was determined using the method of Labarca and Paigen (28).
Modification of culture medium
In some experiments, the pH of culture medium (initially pH 7.7) was titrated with 1 M HCl to pH values between 6.4 and 7.5 after complete equilibration with 5% CO2. To produce macrophage-conditioned medium, purified human macrophages were cultured in 96-well plates at 2 x 105 cells/well in 200 µl complete medium. After 7 days, the medium was removed, centrifuged at 600 x g for 10 min to remove residual cells, titrated to pH 7.5 by the addition of 1 N NaOH, filtered through a 0.22-µm pore size membrane (Millex; Millipore, Bedford, MA), and stored at -20°C. Medium conditioned by Jurkat cells (TIB-152; American Type Culture Collection (ATCC)) and A549 lung carcinoma cells (CCL-185; ATCC) was produced by similar techniques. Cell number was adjusted such that the pH of the conditioned medium after 7 days of culture was similar to that of macrophage-conditioned medium (pH 6.5–6.6). In some experiments, conditioned medium was evaluated after dialysis (Slide-A-Lyser; Pierce, Rockford, IL) against complete medium without serum, after passage through filtration membranes with 3 or 100 kDa cutoffs (Centriprep concentrator; Amicon, Beverly, MA), in which case both the filtrate and the 10-fold concentrated retentate were tested, or after passage through a 1-ml C18 hydrophobic chromatographic cartridge (Waters, Milford, MA). To evaluate the effect of N-iminomethylornithine hydrochloride (L-NIL; Cayman Chemical, Ann Arbor, MI) on myocobacterial growth, a stock solution containing 4 µg/µl was prepared, and aliquots were added to the cultures to produce a final concentration of 5–125 µg/ml. Solutions were prepared contemporaneously from anhydrous solid at the time of addition.
Statistical methods
All results are expressed as mean ± SD unless otherwise indicated. Comparisons between groups were made by ANOVA. Posttest comparisons (performed only if p < 0.05) were made using the Newman-Keuls test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes were cultured for 3 days in 96-well plates, infected
with a suspension of M. bovis BCG, and mycobacterial number
was assessed 7 days after infection based on the measurement of
luciferase activity expressed by the mycobacterial reporter strain.
Mycobacterial growth was highly dependent on the number of human
monocytes present in the cultures (Fig. 1
A). In the absence of
monocytes, little mycobacterial growth was observed. In cultures
containing <5 x 103 macrophages/well, the
viability of the heavily infected cells was poor. Thus, most
mycobacteria were extracellular, because either they were deposited in
the spaces between cells or released from dying cells, and no
significant growth was seen. Mycobacterial growth was first observed in
cultures containing 1 x 104 monocytes, and
increased progressively as monocyte number was increased. Maximal
mycobacterial growth was always observed in cultures containing either
2 x 104 (n = 3) or 5
x 104 cells/well (n = 5) in the
eight experiments in which these two concentrations were compared. In
cultures containing more monocytes, however, mycobacterial growth was
partially (1 x 105 cells/well) or almost
totally suppressed (2 x 105 cells/well) in
all experiments.
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B). In cultures containing 5 x
104 cells/well, progressive mycobacterial growth
was observed, resulting in a 15- ± 5-fold increase in RLU between 1
and 7 days. For cultures containing 2 x 105
cells/well, modest mycobacterial growth was observed at day 4 (2.5- ±
0.7-fold increase in RLU). Between days 4 and 7, however, luciferase
activity decreased in six of nine experiments, resulting in an overall
1.4- ± 0.4-fold increase in RLU between 1 and 7 days. When
mycobacteria were cultured in the absence of macrophages, no net growth
was seen at 7 days.
To confirm the important effect of macrophage density on
mycobacterial growth, a second technique was used to evaluate
mycobacterial number. When mycobacterial growth in cultures containing
5 x 104 and 2 x
105 cells/well was assessed by the
measurement of CFU, the results were comparable to those obtained using
the luciferase assay (Fig. 2). When
mycobacteria were cultured in the absence of macrophages, the
number of CFU decreased progressively in all four experiments (data not
shown).
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When cultures were infected for 24 h and washed three times before measuring luciferase activity, RLU in washed cultures represented 76 ± 13% (5 x 104 cells/well) and 74 ± 11% (2 x 105 cells/well) of the activity detected in unwashed cultures (n = 6, difference not significant). Furthermore, after washing, total RLU at day 1 were similar for cultures containing 5 x 104 and 2 x 105 cells/well. Thus, as previously described (20), most mycobacteria were cell associated in these cultures, and the subsequent differences in mycobacterial growth could not be explained by reduced uptake of mycobacteria in cultures containing 2 x 105 cells/well.
In experiments described thus far, cultures were infected with the same
quantity of mycobacteria, irrespective of cell number. Therefore, it
was important to demonstrate that the 4-fold increase in the number of
mycobacteria/cell (MOI) found when comparing cultures containing 5
x 104 and 2 x 105
cells/well did not explain the differences in mycobacterial growth
observed. We found, however, that despite varying the MOI over a
16-fold range, cultures containing 5 x 104
cells/well were permissive for mycobacterial growth, whereas cultures
containing 2 x 105 cells/well were able to
control mycobacterial growth (Fig. 3).
Thus, when the number of mycobacteria used to infect cultures
containing 2 x 105 cells/well was increased
4-fold compared with that used to infect cultures containing 5 x
104 cells/well, resulting in the same MOI for
these cultures, high density cultures, but not low density cultures,
could still control mycobacterial growth (compare solid bars in Fig. 3). Similarly, when the number of mycobacteria used to infect cells
containing 5 x 104 cells/well was decreased
4-fold compared with that used to infect cultures containing 2 x
105 cells/well, low density cultures were still
permissive for mycobacterial growth (compare hatched bars in
Fig. 3), again indicating that the effect of cell number on
mycobacterial growth is independent of MOI.
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Macrophages cultured at 2 x 105
cells/well progressively acidified the culture medium, an effect that
occurred to a much lesser extent when cells were cultured at 5 x
104 cells/well (Fig. 4A). To evaluate whether such
acidification of the extracellular medium could contribute to the
ability of macrophages to control mycobacterial proliferation, cultures
containing 5 x 104 cells/well were infected
for 24 h, after which the culture medium was replaced with medium
previously adjusted to pH values between 6.8 and 7.7. For cells
cultured initially at pH 6.8, the pH after 7 days was as low as that
observed in cultures containing 2 x 105
cells/well (Fig. 4B). Nevertheless, acidification of the
extracellular medium had little impact on the growth of mycobacteria at
4 or 7 days in macrophages cultured at 5 x
104 cells/well, whereas mycobacterial growth was
controlled in cultures containing 2 x 105
cells/well. Thus, changes in the pH of the extracellular medium could
not account for the differences in mycobacterial growth in low and high
density cultures.
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When monocytes were plated at 5 x 104
cells/well, the cells rapidly formed a monolayer in which most cells
were firmly attached to the surface. After infection, most cells
remained adherent, although a small number of cell clusters were
observed. The cells progressively increased in size, producing an
essentially confluent monolayer at 7 days (Fig. 5A). At this time, essentially
100% of the cells were viable as assessed by their ability to exclude
propidium iodide. After fixation and staining, small clusters of
mycobacteria could be observed in most cells 7 days after infection
(Fig. 5B). Although not quantified, the number of
mycobacteria per cell clearly increased during the week after
infection.
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C).
Despite the low pH of the extracellular medium, >90% of cells in high
density cultures remained viable after 7 days of culture, although
small numbers of dead cells were always present (orange nuclei in Fig. 5C). Subsequently, the viability of high density cultures
declined, and at 10 days numerous dead cells were present. Cells with
condensed nuclei typical of apoptotic cells were observed but were
never abundant; very few nonadherent cells were observed in these
cultures. To evaluate the possibility that dead cells were being lost
from the cultures through ingestion and degradation by the remaining
viable macrophages, the DNA content of adherent cells was measured
after various times in culture. No significant change in DNA content
was observed over the 7-day period in cultures containing 2 x
105 cells/well (day 0, 1.5 ± 0.5; day 7,
1.3 ± 0.6 µg DNA/well; n = 4; p
> 0.05 by paired t test).
After fixation, mycobacteria were observed in high density cultures. In
particular, individual mycobacteria or small aggregates were observed
in the perinuclear region of some, but not all, giant cells (Fig. 5D, arrows). Isolated macrophages containing aggregates of
mycobacteria, as observed in cultures plated at 5 x
104 cells/well, were infrequent. Overall, the
number of mycobacteria per high power field was clearly reduced in high
density cultures. The visualization of mycobacteria required fixation
of the cells, and the viability of the intracellular mycobacteria could
not be assessed.
Effect of macrophage-conditioned medium on mycobacterial growth
The impaired mycobacterial growth in high density cultures could
result from either the production of factors by macrophages that
improve their mycobactericidal activity or the depletion of metabolites
necessary for mycobacterial growth. To test these possibilities,
macrophages were cultured at 2 x 105
cells/well for 7 days, and the conditioned medium was removed,
neutralized to pH 7.5, and added to cultures containing 5 x
104 macrophages/well 1 day after infection. As
shown in Fig. 6A,
mycobacterial growth at 7 days was significantly reduced in cells
maintained in the presence of macrophage-conditioned medium at
dilutions of 1/2 and 1/4 (p < 0.01 and
p < 0.05, respectively) compared with that observed in
cells cultured in unconditioned medium. The inhibitory activities of
conditioned medium from infected and uninfected macrophages were
similar (data not shown). Although mycobacterial growth was reduced in
cells cultured with macrophage-conditioned medium for 7 days,
significant proliferation did occur and was always greater than that
observed in cultures containing macrophages at high density (percent
inhibition of RLU relative to cultures containing 5 x
104 macrophages: 5 x
104 macrophages receiving conditioned medium on
day 1, 68 ± 9%; 2 x 105 macrophages,
93 ± 2%; n = 5, p < 0.01). The
effect observed with conditioned medium was not increased by addition
on two occasions (percent inhibition of RLU: 5 x
104 macrophages receiving conditioned medium on
days 1 and 4, 62 ± 26%; 2 x 105
macrophages, 93 ± 3%; n = 2). Conditioned medium
did not influence the growth of mycobacteria cultured in the absence of
monocytes at 4 or 7 days (p > 0.4 for both
comparisons, n = 3).
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Heating of conditioned medium or passage through a
C18 hydrophobic chromatography column did not
reduce the inhibitory activity. The use of fresh medium that had been
titrated to pH 6.5 using dilute HCl and then neutralized to pH 7.4 did
not reduce mycobacterial growth.
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To evaluate whether the suppressive effect on mycobacterial growth was
a unique property of macrophage-conditioned medium, medium conditioned
by other cell types was also evaluated. Jurkat cells and A549 carcinoma
cells were grown in complete medium until the pH was equivalent to that
of macrophage-conditioned medium, the medium was removed and
neutralized to pH 7.5, and the effects of these conditioned media were
compared (Fig. 6B). Although macrophage-conditioned medium
was somewhat more inhibitory, medium conditioned by the other cell
types also decreased mycobacterial growth at 7 days, and the
differences in inhibition comparing the three media were not
significantly different (n = 4, p >
0.05). As above, high density macrophages had a significantly greater
inhibitory effect on mycobacterial growth than low density macrophages
cultured with conditioned medium.
Effect of L-NIL on mycobacterial growth in macrophages cultured at high density
To evaluate whether NO production was required for the improved mycobacteriostatic activity of macrophages cultured at high density, the effect of the L-NIL, an inducible NO synthase inhibitor, was evaluated. The addition of 5 µg/ml L-NIL on the day of infection did not increase mycobacterial growth at 7 days (RLU/well at 7 days: control, 3870 ± 1326; L-NIL, 4140 ± 1878 RLU/well; p > 0.2). Increasing the concentration of L-NIL to 125 µg/ml or adding 5 µg/ml L-NIL every 48 h after infection also failed to increase mycobacterial growth (data not shown).
Effect of macrophage maturity on the control of mycobacterial growth
In the experiments described thus far, monocytes were infected
after 3 days of culture in vitro. To determine whether the maturation
of these cells influenced the relationship between cell number and
their ability to control mycobacterial growth, monocytes from the same
individuals cultured for 3 and 10 days before infection were compared.
As described above, monocytes plated at 2 x
104 and 5 x 104
cells/well, but not cells plated at 2 x 105
cells/well, were permissive for mycobacterial growth (Fig. 7A). Monocytes cultured for 10
days before infection exhibited progressive differentiation into large
macrophage-like cells that were firmly adherent to the culture wells.
Interestingly, when cultured at 2 x 104
cells/well, these macrophage-like cells were still permissive for
mycobacterial growth, but cultures containing 5 x
104 differentiated macrophages controlled
mycobacterial growth as well as cultures containing 2 x
105 monocytes infected 3 days after isolation
(p > 0.2 at 4 and 7 days comparing the two
populations shown in Fig. 7, A and B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of a number of possible mechanisms through which changes in cell density could influence mycobactericidal activity were investigated. Cell density did not change mycobacterial uptake by the cultured macrophages. Furthermore, the effect of macrophage density on mycobacterial growth was insensitive to the multiplicity of infection over a 16-fold range. Thus, neither of these factors could explain the observed effect.
The control of intraphagosomal pH is also thought to be an important determinant of mycobacterial survival in macrophages. Mycobacterial growth can be directly influenced by an acidic environment, and acidification can increase their sensitivity to oxidant-mediated killing (31, 32). Virulent mycobacteria have been reported to inhibit the acidification of phagosomes through exclusion of vesicular proton-ATPase complexes (33). Acidification of the extracellular milieu can also decrease the intracellular pH of macrophages (34), and we found that macrophages cultured at high density did acidify the extracellular medium to a pH as low as 6.5 after 7 days. Several observations suggested, however, that this change did not account for improved mycobacteriostasis. First, despite acidification of the culture medium to similar levels, macrophages cultured at lower density remained permissive for mycobactericidal growth. In addition, the viability of high density macrophages remained excellent despite the low extracellular pH, suggesting that these cells maintained a physiologic intracellular pH via energy-dependent transport mechanisms. Similarly, the bactericiostatic activity of macrophages cultured at high density could not be attributed to the preferential generation of NO by these cells, because treatment with the inducible NO synthase inhibitor L-NIL did not influence their ability to control mycobacterial replication. Consistent with these observations, prior studies have shown that although human macrophages express inducible NO synthase after mycobacterial infection (35, 36), the levels of NO produced are low, and the inhibition of NO production by human macrophages does not reproducibly increase mycobacterial growth (36, 37). Increased apoptosis of macrophages infected with mycobacteria has been described and in some but not all cases has been associated with mycobacterial killing (38, 39, 40, 41). In this study, the viability of macrophages cultured at high density was excellent at 7 days, few detached cells were present, and the DNA content of the cultures was stable during the 7-day culture period. These findings indicate that macrophage apoptosis or ingestion of infected apoptotic cells by neighboring uninfected macrophages is unlikely to account for the reduced mycobacterial growth seen in high density cultures.
Two observations in our study suggested that the differentiated state of macrophages have an important bearing on their ability to control mycobacterial infection and that both the time in culture and the density of cells present could influence this differentiation process. The mycobacteriostatic activity of both freshly isolated monocytes and macrophages that had been cultured for 10 days before infection was dependent on the number of cells present in the cultures, but fewer of the more differentiated macrophage-like cells were required to control mycobacterial proliferation. Prior studies have shown that the maturation in vitro of human monocytes before infection can modify their capacity to control mycobacterial replication. In these studies, however, an increase (42), a progressive decrease (43), or an initial decrease followed by an increase (44) in the permissiveness for mycobacterial infection has been observed as a function of the time in culture. The important effect of cell density was not evaluated in these studies and may account, at least in part, for the discrepant findings. We also observed that the morphologic features of monocytes cultured at high density were strikingly different from those of cells cultured at low density. In high density cultures, macrophages were larger, cell aggregates were prominent during the first few days of culture, and later times the progressive appearance of giant cells was observed. These changes occurred to a similar extent in uninfected cultures, indicating that mycobacterial infection was not responsible. Byrd (45) previously observed an association between giant cell formation and the ability of cultured monocytes to inhibit mycobacterial proliferation. As in his study, we observed that mycobacteria accumulated in the perinuclear area of giant cells, which may restrict the invasion of adjacent cells (45). A number of signals have been described that increase giant cell formation in cultured macrophages (46, 47, 48). Conditioned medium from high density macrophage cultures did not induce giant cell formation in low density cultures, suggesting that paracrine stimulation by cytokines was not sufficient for the induction of giant cell formation seen in these studies. Culture of cells at high density resulted in the early formation of cell aggregates, compatible with the idea that prolonged membrane contact may have been important.
It should be emphasized that the relationship between increased mycobacteriostatic activity and macrophage differentiation observed in these studies does not prove that the processes are causally related. Furthermore, giant cell formation was not a feature of either low density cultures treated with conditioned medium or macrophages cultured at 5 x 104 cells/well for 10 days before infection, although these populations were able, respectively, to partially or completely inhibit mycobacterial replication. Thus, this type of differentiation, although possibly important, was not indispensable for improved mycobacteriostatic activity. Future studies evaluating the effect of inhibiting macrophage differentiation on mycobacteriostatic activity may prove informative. In this regard, we have recently found that when monocytes are cultured with type I IFNs, they persist as small "monocyte-like" cells for up to 7 days. Under these conditions, monocytes are very permissive to mycobacterial growth, even when cultured at high density (F. Bouchonnet, N. Boechat, M. Bonay, M. Vokurka, and A. J. Hance, manuscript in preparation).
An interesting finding in the current studies was the observation that when low density macrophages were maintained in the presence of medium conditioned by high density macrophages, intracellular mycobacterial growth was reduced. This finding is compatible with the possibility that high density cultures release factor(s) that can stimulate mycobacteriostatic activity or deplete metabolites necessary for mycobacterial growth. The observation that medium conditioned by other cell types also decreased mycobacterial growth in low density macrophages suggests that this effect is not specific for macrophages. Furthermore, modification of the extracellular milieu did not appear to completely explain the increased mycobacteriostatic activity of cells cultured at high density, because low density macrophages cultured in conditioned medium inhibited mycobacterial growth less well than macrophages cultured at high density, and cells cultured in conditioned medium, unlike high density cultures, did not inhibit mycobacterial growth at 4 days.
The ability of conditioned medium to enhance mycobacteriostatic
activity was not decreased by heating, passage through a membrane with
a 3-kDa cutoff or passage through a C18
hydrophobic column, whereas the activity was completely lost by
dialysis. In addition, the 10-fold concentrated retentate obtained by
ultrafiltration of conditioned medium did not contain increased
activity. These findings argue strongly against a role for soluble
secreted proteins or lipid mediators in this phenomenon. Thus, although
cultured macrophages are secrete large quantities of cytokines,
including IFN-, TNF-, and IL-12 (22, 23, 30, 49, 50), our results are compatible with prior studies indicating
that these cytokines are not sufficient to induce strong
mycobactericidal activity in otherwise permissive human macrophages.
Low molecular mass substances produced by macrophages, and other cell
types can modify the activity of macrophages and improve their
resistance to intracellular pathogens, (e.g., ATP and nonheme iron)
(20, 51), and the release of such agents could explain our
findings. Attempts to isolate a putative stimulatory factor by ion
exchange chromatography were unsuccessful, but the buffers required for
these studies proved to be toxic for macrophages, which complicated
this endeavor (data not shown). Alternatively, it is the possibility
that substances that deactivate macrophages or that are required for
optimal mycobacterial growth had been depleted by the metabolically
active cells (e.g., iron, adenosine, amino acids)
(52, 53, 54, 55, 56). The activity of conditioned medium was
completely lost after dialysis against serum-free medium, indicating
that the culture medium, not human serum, would have to be the source
of such a metabolite. Furthermore, because conditioned medium diluted
4-fold with fresh medium retained some activity, this metabolite would
have to be present in suboptimal amounts in fresh medium. Further
studies will be required to identify the substances responsible for the
effects observed with conditioned medium and to determine whether
macrophages within granulomas can "condition" their environment in
a similar fashion.
Taken together, these results indicate that the ability of human macrophages to control mycobacterial infection in vitro is strongly influenced by the density of cells present and indicate that cell density and the time in culture before infection can influence the differentiated state of these cells and their capacity to modify the external milieu. The requirement for both monocytes and more mature macrophages to be present at a critical density to suppress mycobacterial proliferation offer insights into why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections. The availability of this model, in which the same cells are either permissive or resistant to mycobacterial infection, should be useful in further characterizing the cellular events that are indispensable for the development of mycobactericidal activity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Allan J. Hance, Institut National de la Santé et de la Recherche Médicale Unité 552, IMEA-Institut National de la Santé et de la Recherche Médicale, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, 75018 Paris, France.
3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; L-NIL, N-iminomethylornithine hydrochloride; MOI, multiplicity of infection; RLU, relative light units.
Received for publication October 16, 2000. Accepted for publication March 8, 2001.
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, yet succumb to tuberculosis. J. Immunol. 162:5407. in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249. gene-disrupted mice. J. Exp. Med. 178:2243. receptor have profoundly altered responses to infection with bacillus Calmette-Guérin and subsequent challenge with lipopolysaccharide. J. Exp. Med. 178:1435. is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561.[Medline]
-receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335:1941.-receptor deficiency in an infant with fatal bacille Calmette-Guérin infection. N. Engl. J. Med. 335:1956. interferon, interleukin-4, and tumor necrosis factor. Infect. Immun. 58:2675. (TNF) promotes growth of virulent Mycobacterium tuberculosis in human monocytes. Iron-mediated growth suppression is correlated with decreased release of TNF from iron-treated infected monocytes. J. Clin. Invest. 99:2518.[Medline]
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/IL-3 is associated with restriction of virulent Mycobacterium tuberculosis cell to cell invasion in human monocyte monolayers. Cell. Immunol. 188:89.[Medline]
: comparison with other stimulating factors. Am. J. Respir. Cell Mol. Biol. 6:57.
up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation. J. Immunol. 162:3607. blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cell to degrade tryptophan. Proc. Natl. Adad. Sci. USA 81:908., indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J. 5:2516.[Abstract]
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), prepared as described above, or no
monocytes (
) were infected with M. bovis BCG reporter
strain, and luciferase activity was measured after the indicated time
in culture. Results are the mean ± SD for nine independent
experiments performed using cells obtained from different
individuals.
), prepared as described
above, were infected with the M. bovis BCG reporter
strain. After the indicated time in culture, the number of mycobacteria
present were assessed by measuring luciferase activity (RLU, 
, 
, 
)
or 2 x 105 cells/well (