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Laboratory of Bacterial Pathogenesis, Kuzell Institute for Arthritis and Infectious Diseases, California Pacific Medical Center Research Institute, San Francisco, California
Correspondence: Dr. Luiz E. Bermudez, Kuzell Institute, 2200 Webster Street, Suite 305, San Francisco, CA 94115. E-mail: luizb{at}cooper.cpmc.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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prior to infection with M. avium resulted in
greater production of IL-12 compared with unstimulated macrophages.
Culture supernatant of M. avium-infected macrophage
monolayers, but not control macrophages, partially inhibited IL-12
production by IFN--stimulated macrophages. This partial inhibition
was not reversed by antiinterleukin-10 (anti-IL-10) and
antitransforming growth factor ß1 (anti-TGFß1)-neutralizing
antibodies. M. avium infection of macrophages in vitro also
suppressed IL-12 synthesis induced by Listeria
monocytogenes infection. Immunohistochemistry staining of spleen
of infected mice showed that IL-12 production by splenic macrophages
was more pronounced in the beginning of the infection but decreased
later. Our data indicate that M. avium infection of
macrophages suppresses IL-12 production by infected cells and that the
suppression was not a result of the presence of IL-10 and TGFß1 in
the culture supernatant. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The host’s immune response to M. avium infection has been
shown to be complex and to involve the participation of a number of
cells, such as CD4 T cells [4
, 5
], natural
killer (NK) cells [6
, 7
], mononuclear
phagocytes [8
, 9
], and perhaps 
T
cells [10
]. Recent studies in vitro and in vivo have
demonstrated that the cytokine interleukin-12 (IL-12) plays an
important role in the orchestration of the immune defense of the host
against M. avium, and deficiency of the cytokine is
associated with accelerated progression of the disease
[11
12
13
14
]. Furthermore, individuals with a genetic
deficiency of functional IL-12 receptors have an increased
predisposition for mycobacterial infections [15
]. IL-12
participates in the host defense against Mycobacterium
tuberculosis as well. For example, IL-12 is secreted by
macrophages following infection with M. tuberculosis in
vitro [16
, 17
], and work carried out in
mice supports a key role for IL-12 in the mechanisms of immunity to
M. tuberculosis [18
, 19
].
Previous results by this as well as other laboratories
[20
21
22
] have shown that one of the mechanisms that
pathogens use to survive intracellularly is to shift the pattern of
cytokine production by infected cells to their own benefit. Therefore,
it is possible that synthesis of important cytokines such as IL-12 is
impaired in M. avium infection. To investigate this
possibility, we examined if M. avium infection of human
macrophage monolayers in vitro triggered IL-12 production; if infected
macrophages in the monolayer synthesized IL-12; if stimulation of
macrophages with interferon-gamma (IFN-) was associated with changes
in the pattern of IL-12 production; that M. avium infection
has a suppressive effect on IL-12 production induced by macrophage
infection with Listeria monocytogenes; and if IL-12
production was impaired in vivo.
Our results suggest that M. avium infection triggers IL-12 synthesis by phagocytic cells, but simultaneously, with the progression of the infection, IL-12 production by infected macrophages decreases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L. monocytogenes 2776 is a clinical isolate (blood). It was cultured on blood agar prior to the assays. Viability was also determined by the LIVE-DEAD assay (Molecular Probes).
M. avium sonicate was prepared by sonicating 1 x 104 M. avium 101 for 8 min on ice (20 watts). For the experiments, 50 µl sonicated preparation was used.
Isolation of the macrophages
Venous blood from five healthy volunteers was drawn into
heparinized syringes, layered onto Ficoll-Hypaque (Sigma Chemical Co.,
St. Louis, MO), and centrifuged for 40 min at 1500 rpm (250
g). Peripheral blood mononuclear cells were isolated from
the interface and washed three times in RPMI-1640 (Sigma Chemical Co.).
Approximately 5 x 105 monocytes were then placed per
well in a 24-well tissue-culture plate (Costar, Cambridge, MA) and
incubated for 1 h to allow for attachment. After 1 h at
37°C, nonadherent cells were removed by three washes with warm
(37°C) HBSS. Mononuclear phagocytes cultured for 4 days developed
morphologic characteristics of macrophages. The cells (40%–50%),
initially added per well, detached after 10 days of culture in the
control as well as in the experimental groups. Viability of the
monolayers was determined by using trypan blue as previously described
[14
]. Only monolayers with >95% of viable cells were
used for the described experiments. For experiments using microscopy,
monocytes (105 cells) were seeded per well in 8- or 2-well
Lab-Tek slides (Nunc, Naperville, IL) as previously described
[20
].
Infection of the monolayers
Macrophage monolayers (1x105 cells) were infected
with M. avium or M. smegmatis at a ratio of 10
bacteria:1 cell or 1 bacteria:1 cell. Infection was allowed to occur
for 2 h, after which extracellular bacteria were removed by
washing [14
]. Some monolayers were then lysed to
establish the number of intracellular bacteria. In some experiments,
macrophage monolayers were lysed after 1, 2, 3, and 5 days to
quantitate viable intracellular bacteria. Culture supernatant was
obtained following 24 and 48 h infection. Infection with L.
monocytogenes was carried out using a ratio of 10 bacteria:1
macrophage and was allowed to occur for 2 h before washing
extracellular bacteria. In some assays, macrophages were infected with
M. avium for 24 h and 72 h and then exposed to
L. monocytogenes for 24 h.
IL-12 protein measurement
IL-12 was measured in tissue-cultured supernatant. Supernatants
were obtained from macrophage cultures 0, 1, 2, 3, and 5 days after
infection. Samples were clarified through a 0.2 µm pore-sized filter
(Gelman Sciences, Ann Arbor, MI) and stored at -70°C until used.
IL-12 concentration was determined by enzyme-linked immunosorbent assay
(ELISA; Biosource, Camarillo, CA) by measuring the active p70 protein
according to directions of the manufacturer. The limit of detection of
the assay was 10 pg/ml.
Reagents
Monoclonal antibody (mAb) to human IL-12 was purchased from R &
D Systems (Minneapolis, MN). The antibody is specific for IL-12 and
does not bind to any other cytokines. Lipopolysaccharide from
Escherichia coli 0111B6 was purchased from List Biological
Laboratories (Campbell, CA). Anti-mouse antibody labeled with Texas-Red
or fluorescein was purchased from Amersham Life Science (Arlington
Heights, IL). Recombinant human IFN- was kindly provided by
Genentech (South San Francisco, CA). It had an activity of
107 U/mg protein. The cytokine was prepared in buffer
containing 10% albumin, aliquoted in small volumes, and kept frozen
before use. Mouse anti-human IL-10 neutralizing antibody immunoglobulin
G (IgG)2a was purchased from Biosource. Antibody (1 ng) was
able to neutralize 104 U cytokine. An IgG2a was
used as nonrelevant control. Rabbit anti-human transforming growth
factor (TGF)-ß neutralizing antibody was obtained from R & D Systems.
Rabbit anti-human antibody (antiactin) was used as nonrelevant control.
Staining procedures
Production of IL-12 (p70) by infected and uninfected macrophages
was determined by standard staining procedures [23
].
Briefly, macrophage monolayers seeded in chamber slides were infected
with 1:1 or 10:1 bacteria:cell ratio in the presence of brefeldin A
(BFA; 10 µg/ml). After 2 h, extracellular bacteria were removed
by extensive washing with HBSS. Fresh medium was added, and infection
was followed for 4 h, 24 h, 48 h, or 72 h. The
chambers were then fixed with 2% paraformaldehyde, pH 7.2, for 1 h at room temperature. After washing, cells were permealized in 0.1%
Triton-X 100 for 15 min at room temperature. Control monolayers with
uninfected macrophages were treated with BFA (same period and
concentration), and no toxic effect was observed (unpublished results).
Slide chambers were then washed and incubated with 1% fetal bovine
serum for 30 min at room temperature. After removal of the serum, mouse
anti-human IL-12 antibody was added for 1 h at room temperature.
Following removal of the first antibody and washing, a second antibody,
Texas-Red-conjugated goat anti-mouse antibody, was added for an
additional hour. Controls with irrelevant primary antibody (mouse
antilipid A) and using the second antibody alone were run in parallel.
The cells were then washed, mounted in mounting medium, and sealed.
Slides were observed under a fluorescence Nikon microscope. The
intensity of the staining was scored blindly by two individuals.
Infection of mice with M. avium and immunohistochemistry: C57 BL/6 female mice (20 g) were infected intravenously (i.v.) with M. avium strain 101, and at 48 h and 2 weeks after infection, spleens were aseptically removed and fixed with 2% formaldehyde as described [25 ]. Fixed material was then stained with rabbit anti-mouse IL-12 antibody as previously described [25 , 26 ] using a vector kit (Vector Laboratories, Burlingame, CA).
Statistical analysis
Comparisons between control and experimental groups at the same
timepoint were analyzed using the Student’s t-test. Each
experiment was performed at least five times, and the results represent
the mean ± SD of the data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Staining of macrophages for IL-12
To examine if IL-12 released in the supernatant of M.
avium- and M. smegmatis-infected monolayers was
produced by infected or uninfected macrophages (or both populations),
monolayers were infected with 10:1 and 1:1 bacteria:cell ratios, and
cells were stained using imunofluorescence for IL-12 at 4, 24, 48, and
72 h. As observed in Figure 1
at 24 h, both populations of cells (infected and uninfected)
are shown to produce IL-12, although it was suggested that most of the
IL-12 is synthesized by uninfected cells. However, at 48 h, it was
clear that in the M. avium-infected monolayer, the majority
of the IL-12 was produced by uninfected cells. Table 3
and Figure 1A
and 1B
, show the quantitation of IL-12 production
by infected and uninfected macrophages. IL-12 was not observed in
macrophages infected for 72 h.
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would result in a change in the pattern of IL-12 secretion by
the M. avium-infected monolayer, macrophages were treated
with recombinant IFN- (102 U/ml) in combination with
M. avium sonicate for 24 h prior to and following
M. avium infection. Pretreatment with IFN- did not
influence phagocytosis of M. avium [9
].
Although IFN--treated macrophages had 3.8 ± 0.4 x
105 intracellular bacteria, control monolayers had 2.7 ± 0.3 x 105 bacteria (P>0.05).
Supernatants were then obtained, and the concentration of IL-12 was
determined. It was seen that when macrophages were stimulated with
IFN- plus M. avium sonicate prior to infection, the
concentration of IL-12 in the supernatant increased significantly after
24 h (Fig. 2
). However, if the macrophage monolayer was infected with M.
avium for 24 h, the increase in IL-12 secretion, although
greater than infected untreated control, was significantly lower than
the production of IL-12 observed in monolayers stimulated prior to
infection (Fig. 2)
.
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and sonicate of 103 M. avium in 50 µl
for 24 h in the presence or absence of 0.3 ml culture supernatant
(added 24 h before stimulation) obtained from infected monolayers
(24 and 48 h). As shown in Figure 3
, the presence of 24 h and 48 h supernatant resulted in
partial inhibition of IL-12 production by IFN--treated macrophages.
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Table 5 shows that M. avium suppresses L. monocytogenes-induced IL-12 production in macrophages and that suppression is more accentuated after 72 h rather than 24 h of M. avium infection.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based on the studies by Castro and colleagues [11 ] and Saunders and colleagues [12 ], it is clear that in vivo IL-12 production leads to protective immunity against M. avium, and neutralization of IL-12 with antibodies resulted in the inability of BALB/c mice to control the infection.
IL-12 acts on precursor T cells promoting their differentiation into a
TH1 phenotype (responsible for IFN- secretion) and on NK
cells, increasing their ability to proliferate and to secrete cytokines
[28
]. Using different study designs, several
laboratories have shown recently that IL-12 has an important role in
the host defense against pathogenic mycobacteria
[11
12
13
14
, 16
, 17
] and that
infection in vitro of macrophages with M. tuberculosis
resulted in IL-12 secretion [16
, 17
].
Our results using human monocyte-derived macrophages and three AIDS
isolates of M. avium confirmed the observation obtained with
M. tuberculosis [16
, 17
] showing
that infection of macrophage monolayer resulted in IL-12 production.
However, we also showed that the production of IL-12 is primarily
derived from uninfected macrophages and not from infected cells; the
production of IL-12 has an inverse relationship with the period of
infection as well as with the number of viable intracellular bacteria;
macrophage monolayers stimulated with IFN- secreted an increased
amount of IL-12, some of which was produced by M.
avium-infected macrophages; supernatant from M.
avium-infected macrophage monolayers suppressed IL-12 production
by uninfected macrophage stimulated with IFN- plus M.
avium sonicate; and the inhibitory effect of supernatant on IL-12
production was IL-10- and TGFß1-independent.
Alternatively, all the data could be interpreted by the effect of mycobacterial products of IL-12 production, which is dose-related with a sharp peak and a dramatic fall. However, the results with M. smegmatis do not support this conclusion (Table 2) .
The observation that M. avium-infected macrophages are not responsible for the production of IL-12 detected in culture supernatants supports previous work showing that macrophages infected with M. avium become refractory to stimulation of several cytokines, impairing their ability to inhibit or kill intracellular bacteria 2–3 days after infection [29 ]. Studies by Zhang and colleagues [30 ] have demonstrated the production of IL-12 in vivo in tuberculosis pleuritis, a human model of anti-M. tuberculosis immune response. In addition, Fulton and colleagues [16 ] showed production of IL-12 by monocyte monolayers when infected with M. tuberculosis, however it is unknown if IL-12 was synthesized by infected or uninfected macrophages. Our findings that IL-12 p70 was induced approximately 4 h after infection of the monolayer with M. avium suggests (but is not conclusive) that IL-12 can be, at least initially, produced by macrophages that ingested M. tuberculosis (because the period of time between infection and IL-12 production is too short to be explained by a secondary stimulation leading to IL-12 synthesis by uninfected cells). Results obtained with mouse bone marrow macrophages [18 ] seem to support our findings that IL-12 concentration peaks early in the infection and declines afterward. Measure of IL-12 at 4 h postinfection suggests that M. avium-infected macrophages produce IL-12 soon after infection, but it was of short duration. The M. avium-mediated inhibition of IL-12 production can be, at least, a partial explanation for a previous observation that a TH2 type of CD4 T-cell response is present only after weeks of M. avium infection in mice [31 ].
Treatment of macrophage monolayers with IFN- up-regulated the
synthesis of IL-12 by infected and uninfected macrophages. However,
infected macrophages had an impaired ability to respond to IFN-
compared with uninfected ones. Similar findings of impaired response to
cytokines have been demonstrated previously regarding stimulation of
infected macrophages with tumor necrosis factor-alpha (TNF-
)
[20
] and were almost completely reversed by the presence
in the supernatant of neutralizing antibody to TGFß1
[20
].
Our data also show that M. avium-dependent suppression of IL-12 production can be reproduced in part by using culture supernatant of infected macrophages, suggesting that secreted factors have a role in the inhibition of IL-12 production. We also showed that the inhibitory effect of culture supernatant was not reversed by using neutralizing anti-IL-10 or anti-TGFß1 antibodies. IL-10 and TGFß1 have been shown to be synthesized and secreted by macrophages infected with M. avium in vitro and in mice [20 , 21 ]. Recent studies by Takenaka and colleagues [32 ] as well as a study by Bogdan and Nathan [33 ] have demonstrated that IL-10 suppresses macrophage function, including the production of IL-12. In addition, a recent paper by Weinheber and colleagues [34 ] demonstrates that Leishmania mexicana amastigotes block IL-12 production by macrophages, although the suppression is also not reversed by the use of neutralizing anti-IL-10 antibody. Our data agree with recent observations that mycobacteria-infected macrophages secrete bacterial products (lipids among them) that can inhibit the function of bystander macrophages as shown for Mycobacterium bovis bacillus Calmette-Guerin (BCG) by Atkinson and colleagues [35 ] and M. tuberculosis by Beatty and colleagues [36 ].
Work by von Grunberg and Plum [37 ] suggests that isolates from AIDS patients in contrast to environmental isolates produce increased amounts of p40 homodimer, which can be immunosuppressive. Although this observation can probably explain some of our findings, the timeframe of the inhibition seen in our studies is not the same for the production of p40 IL-12, implying that other factors may be present.
Our observation that IL-12 production is decreased in mice infected with M. avium for 2 weeks confirms the observation in vitro.
The importance of this finding is better demonstrated by the M.
avium-induced suppression of IL-12 production when macrophages are
infected with L. monocytogenes. This result indicates that
the M. avium effect on macrophage function is likely to
occur by specific interference in signal transduction as has been shown
for TNF- [20
] and in the case of M.
tuberculosis infection and IFN--mediated stimulation of
macrophages [38
].
The interaction between the host and M. avium is complex. The ability of the host to mount an effective immune response against the bacterium is counteracted by the ability of the bacterium to exploit the immune system triggering the production and secretion of immunosuppressive cytokines. Although macrophages activated prior to M. avium infection appear to be able to control the intracellular multiplication of the bacteria [9 ], an increasing body of data indicates that resting macrophages, once infected, progressively lose their capacity to present antigens [39 ], produce and secrete cytokines [40 , 41 ], and respond to stimulation [29 , 39 ]. The data shown in this study add to the increasing list of observations demonstrating M. avium-mediated down-regulation of the host immune response.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Current address of Sang Kim: Department of Pathology, Kon-Kuk University, Choong-Buk, South Korea.
Current address of Dirk Wagner: Department of Internal Medicine I, University of Tübingen, Tübingen, Germany.
Received August 15, 2001; accepted August 15, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells can both lyse Mycobacterium avium-infected human macrophages and stimulate macrophage-mediated mycobacteriostatic activity Immunol. Infect. Dis. 5,183-188
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