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Production, and Apoptosis in Terminal Infection of Mice with Mycobacterium avium1
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. After initial rapid growth, bacterial
numbers slowly increased from 
107 at 6 wk to more than
5 x 108 at 28 wk, indicating that the resistance
mechanisms so generated were not adequate to contain the infection.
During infection, apoptosis of both CD4+ and
CD8+ T cells, measured immediately ex vivo by staining with
Annexin V, increased steadily. With some individual exceptions, there
was a close correlation between apoptosis of CD4+ cells and
level of IFN- production by cultured spleen cells. By 34 wk
postinfection, there was an abrupt cessation of IFN- production. No
IL-4 was detected, ruling out a switch to Th2 profile. Subsequently,
bacterial numbers increased still further to >5 x
109 per lung, and the mice lost body weight and would have
died if not killed for experimental or humane reasons. The possibility
that T cells exposed over this prolonged period to extremely high doses
of Ag may become tolerant by a process of terminal differentiation is
discussed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, produced
largely by CD4+ T lymphocytes, and the formation
of granulomas to isolate infectious foci (2, 3). This is
supported by production of other of the proinflammatory cytokines
(4), namely TNF, which is prominent in granuloma formation
(5), and IL-12, which promotes the differentiation of
IFN-- producing cells (6). CD8+ T
cells play a still-controversial role, possibly lysing cells and
killing the bacteria they harbor (7). Recent reports of
patients with multiple intracellular bacterial infections due to
defects in production of, or response to, IFN- (8) and
IL-12 (9, 10) confirm the importance of these type 1
cytokines in resistance of humans to intracellular bacteria.
The question arises as to whether, during the course of a chronic
infection, there are changes in the immune response that limit its
effectiveness. Such changes could include feedback control mechanisms
whose value is to limit the damage due to chronic inflammation
(11). Alternatively, they may be analogous to those
described in viral infection, in which persistence of high viral load
leads to anergy and loss of protective CD8+ T
lymphocytes (12). Evidence of a late loss of effective
immunity to tuberculosis comes indirectly from patients diagnosed with
advanced disease who fail to respond to the tuberculin skin test
(11) and whose T lymphocytes show reduced IFN-
production on culture with mycobacterial Ags (13, 14).
Although a number of explanations have been advanced to explain these
clinical observations (11), an experimental model in which
to study the evolution over time of the immune response to a
mycobacterium would be of particular interest.
Mycobacterium avium, the commonest bacterial complication of
AIDS, is largely nonpathogenic for immunologically intact humans.
However, intranasal infection of mice with M. avium leads to
chronic infection that spreads from the lung to liver and spleen
(15), making it a suitable model in which to study the
changes in the immune response to chronic bacterial infection. IL-12
(16, 17) and IFN--producing CD4+
T lymphocytes (15) are required to activate macrophages
and control the infection. Numbers of bacteria increase steadily for
the first 5–6 wk until these mechanisms come into play and bacterial
proliferation slows. We have observed that, when mice are held
for more than 30 wk, individuals begin to lose weight and become
moribund, in a manner that might be expected of chronic, untreated
tuberculosis. This gave us the opportunity to study the progress of a
mycobacterial infection and changes in the immune response from the
earliest stages of acquired cellular immunity to the onset of the final
decline of the animal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. avium serovar 8 was isolated from an AIDS patient (15) and grown in Middlebrook 7H9 broth (Difco, Detroit, MI) for 7–10 days. It was of smooth transparent colony type. CFU were determined by culturing 10-fold dilutions on Middlebrook 7H11 agar, and the broths were frozen in 1-ml ampoules at -70°C. Before infection of the mice, the bacteria were thawed at 37°C and subjected to sonication for 10 s to disperse clumps. Female 6- to 8-wk-old C57BL/10 mice were pedigree bred and maintained under conventional but infection-free conditions in the animal house of the Department of Microbiology, University of Melbourne (Victoria, Australia). Working under a biosafety hood, the mice were placed under light penthrane anesthetic and 50 µl of the M. avium suspension, adjusted to deliver 105 CFU, placed on their nares to be breathed in smoothly by the mouse. A retrospective dose check was made by culturing diluted samples onto Middlebrook agar.
To assess the course of infection, mice were weighed and sacrificed by CO2 narcosis, and their lungs and livers were removed aseptically. Infected mice were compared with age- and sex-matched normal controls. Organs were individually homogenized in 5 ml of PBS (pH 7.2) using an Ultra Turrax tissue homogenizer (Janke and Kunkel, Bresigau, Germany). Serial 10-fold dilutions of homogenates were prepared in 96-well microtiter plates (Nunc, Riskilde, Denmark). Suitable dilutions were sampled onto Middlebrook plates and incubated at 37°C for 5–6 days. Colonies were counted to determine the number of viable bacteria.
Reagents
Anti-CD4 mAb from GK1.5 hybridoma and anti-CD8 mAb from
3.168 hybridoma cell line (18) were prepared by
precipitation of ascitic fluid with 50% ammonium sulfate. Anti-CD3 mAb
was protein A purified from culture supernatants of hybridoma 145-2C11,
and was the kind gift of Dr. P. Mottram, Royal Melbourne Hospital
Department of Medicine. FITC-conjugated anti-rat Ig was purchased
from Silenus (Melbourne, Australia). Annexin V conjugated to PE,
PE-conjugated anti-mouse CD4 (H129.19), or anti-CD8
(7) mAbs, FITC-conjugated anti-mouse IFN-
(XMG1.2), and PE-conjugated anti-mouse IL-4 mAb (BVD4-1D11) were
all purchased from PharMingen (San Diego, CA). Binding buffer for cell
staining was prepared at 10x concentration as follows: 0.1 M
HEPES/NaOH adjusted to pH 7.4 in 1.4 M NaCl and 25 nM
CaCl2. It was stored at 4°C and diluted 1/10
for use.
Spleen cell preparation
Spleens were aseptically removed from mice killed at different stages of infection. Single-cell suspensions from individual spleens were obtained by teasing through 80-gauge/80-mesh stainless steel sieves. Viable lymphocytes were isolated by Ficoll-Histopaque (Sigma, Castel Hill, NSW, Australia) density-gradient centrifugation. Cells were washed and adjusted to a concentration of 2 x 106 cells/ml in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Life Technologies), 36 mg/L L-asparagine, 116 mg/L L-arginine, and 216 mg/L L-glutamine, 10 mM HEPES, 5 x 10-5 M 2-ME, 60 µg/ml penicillin, and 100 µg/ml streptomycin.
IFN- bioassay
For assay of released IFN-, cells were stimulated with and
without the addition of 5 x 106/ml live
M. avium for 72 h. Supernatants were harvested, and
filtered using a 0.45-µm millipore filter (Minisart, Sartorius,
Gottingen, Germany). IFN- was assayed by its ability to inhibit the
growth of the WEHI-279 cell line (19). Serial 3.15-fold
dilutions of culture supernatants were incubated at 37°C 5%
CO2 with 104 WEHI-279
cells/well in supplemented DMEM/F12 in 96-well flat-bottom microtiter
plates (Nunc, Roskilde, Denmark). All samples were assayed in
triplicate. After 3 to 4 days, cultures were pulsed with 10 µl of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5
mg/ml) for 4–6 h, and then incubated with 100 µl of SDS-0.01 N HCl
overnight. OD was measured at 540 nm with 690 nm as a reference using a
Multiskan microplate reader (Multiskan MCC, Helsinki, Finland). Titers
were read from dose-response curves by comparison with standard. Medium
was included as background. Specificity was checked by incorporating
anti-IFN- mAb in some wells. Neutralization of >90% was
accepted as indicating specificity.
Enzyme-linked immunospot (ELISPOT)3 assay
Spleen cells prepared as described above were assayed for the
frequency of IFN--producing cells by ELISPOT. White, MaxiSorp
96-well plates (Nunc) were coated with anti-IFN- mAb (HB170) by
overnight incubation at 4°C. Cells were added to washed and blocked
plates (2 x 105, 1 x
105, 5 x 104, and
2.5 x 104 cells/well in triplicate) and
incubated with and without 5 x 106/ml live
M. avium for 72 h at 37°C 5%
CO2. Cells were then washed off, and IFN-
bound to the nitrocellulose was detected with biotinylated rat
anti-mouse IFN- mAb (XMG1.2) (PharMingen), followed by
streptavidin-alkaline phosphatase and substrate. Spots were counted by
light microscopy. The frequency of IFN--producing cells was
calculated by averaging the number of spots for triplicate wells.
Intracytoplasmic cytokine (ICC)3 staining
ICC staining was used to determine the frequency of
IFN-- or IL-4-producing
CD4+ and CD8+ T cells
according to previous methods (20). Spleen cells were
transferred to 24-well culture plates (Costar, Corning, Corning, NY) at
a concentration of 2 x 106 cells in 1 ml,
and the T cells were stimulated with protein A-purified immobilized
anti-CD3 mAb (145-2C11) (2.5 µg/ml) (21) in the
presence of 2 µM monensin (PharMingen) for 6 h at
37°C in 5% CO2. Aliquots of 1 x
106 cells were transferred to FACS tubes (Falcon
12 x 75-mm round-bottom tubes; Becton Dickinson, San Jose, CA),
and washed with staining buffer (PBS, 1% heat-inactivated FCS, 0.1%
(w/v) sodium azide). Cells were then stained with either PE-conjugated
anti-mouse CD4 (H129.19) or CD8 (53-6.7) mAb, at a concentration of
2 µg/ml in staining buffer for 30 min on ice. Cells were washed twice
and then fixed and permeabilized in PBS containing 4% paraformaldehyde
and 0.5% saponin for 20 min on ice. Cells were washed twice in
permeabilization buffer (PBS, 0.5% saponin, 1% FCS) and stained with
either 5 µg/ml FITC-conjugated anti-mouse IFN- (XMG1.2), or 2
µg/ml PE-conjugated anti-mouse IL-4 mAb (BVD4-1D11), in
permeabilization buffer for 30 min on ice. Cells were again washed
twice with permeabilization buffer and were resuspended in PBS for
analysis by flow cytometry using Becton Dickinson FACSort and CellQuest
software. Routinely, 30,000 events were collected and analysis gates
were set on lymphocytes according to forward- and side-scatter
properties. In all experiments, unstained cells and cells stained
separately with each fluorochrome were included to optimize
compensation settings. Cytokine-specific signals were determined using
unstained cells or cells stained with the appropriate isotype control.
Results are expressed as the percentage of cytokine-producing cells in
either the total, CD4+, or
CD8+ cell population.
Apoptosis determination
Spleen cells were adjusted to a concentration of 1 x 106/ml of PBS, and 1 ml aliquots were centrifuged in FACS tubes. The cells were surface labeled for 30 min on ice in 200 µl PBS with 10 µg/ml anti-CD4 or anti-CD8 mAb. The cells were washed once with cold PBS and stained on ice for another 30 min with FITC-conjugated anti-rat Ig. Cells were washed again with cold PBS, resuspended in 100 µl binding buffer, and stained with 1.6 µl PE-conjugated Annexin V and 7-AAD (PharMingen) by gently mixing for 15 min at room temperature in the dark. Binding buffer was added to each tube to restore the volume to 300 µl, and the samples were analyzed by FACSort (Becton Dickinson, San Jose, CA). In setting compensation, cells stained with Annexin V alone or 7-AAD alone were used. Apoptotic cells stain with Annexin V, while necrotic cells stain with both Annexin V and 7-AAD (22).
Statistics
The statistical significance of experimental data was determined by Student’s t test. Differences with p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies on the time course of M. avium
infection showed that, following intranasal infection, bacterial
numbers increased steadily until 5–6 wk. At this time, IFN-
production by T lymphocytes became detectable in vitro, after which
bacterial growth slowed, reaching a little over
107 in lungs, and 105 in
spleen and liver during the 15 wk of observation (15).
However, when the mice were held for longer periods, their condition
was noted to deteriorate 30 wk or more postinfection.
To study this phenomenon, female C57BL/10 mice were infected
intranasally with 105 CFU of M.
avium, and bacterial counts were performed at intervals shown in
Fig. 1
, upper panel. Following
an early rapid increase in the lungs and spread to the liver and
spleen, bacterial numbers grew steadily over the entire period of
observation, reaching numbers in excess of 5 x
109 per lung before the mice became moribund
about 35–38 wk after infection.
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, lower panel). When the weight reached less than 80% normal,
mice were euthanized for ethical reasons. Histological observations on
the same mice revealed increasing numbers of granulomas in the lungs
(Fig. 2). By the time the mice were
suffering significant loss of weight, their lungs were almost
completely consolidated.
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production during M. avium infection
Spleen cells recovered at different times postinfection were
cultured with live M. avium as antigenic stimulus, and the
supernatants were harvested 3 days later for bioassay of IFN- (Table I). As previously observed, there was
substantial production of IFN- 6 and 12 wk postinfection
(15). However, when observations were extended, IFN-
recall was dramatically reduced at 38 wk postinfection. To check
numbers of IFN--producing cells, ELISPOT assays were performed and
showed a decline from 140 IFN--producing cells per
106 spleen cells at 6–12 wk to
35/106 at 38 wk. This compared with numbers in
normal mice below the limit of detection
(<5/106). Results were consistent over a number
of replicate experiments.
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mAb, and PE-labeled anti-CD4 or
anti-CD8 mAbs. Fig. 3 shows typical
data from individual mice after compensation for overlap of the two
colors. Although IFN--producing cells in excess of normal can be
seen at 6 and 12 wk postinfection, this had declined to the level seen
in normal mice by 38 wk postinfection. Both CD4+
and CD8+ cells stained for IFN- production,
with no discernible difference in the intensity of staining in the two
subpopulations. A point of interest was the number of IFN--producing
cells at 6 and 12 wk not staining with either anti-CD4 or
anti-CD8 mAb. Because these cells had been stimulated with
anti-CD3 mAb, it must be assumed they were T cells, but whether
/ß T cells that had lost their CD4/CD8 expression, or whether
/
T cells is not yet clear.
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summarizes the data from groups
of three mice individually analyzed per time point. At 6 and 12 wk
postinfection, 1.4% of all spleen cells produced IFN- under these
conditions. At these time points, almost 10% of the
CD4+ cells and 10% of CD8+
cells contained IFN-. This compared with a background of 2.3%
CD4+ and 5% CD8+ cells
from uninfected mice. In repeated experiments, the background for
CD8+ cells was higher than for
CD4+ cells, although this experiment was
unusually high. By 36 wk postinfection, the percentage of
CD4+ and CD8+ producers had
returned to normal. When the absolute numbers of IFN-
producers per spleen were calculated, it can be seen that due to the
decline in numbers of T cells per spleen at 38 wk, the numbers of
IFN- producers were significantly less than in uninfected mice.
Because of the greater numbers of CD4+ cells,
they represented more than 50% of all IFN--producing cells, and
CD8+ cells were less than 20% at all stages of
infection.
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Spleen cells were prepared from mice at various stages of
infection and divided on the basis of FACS analysis (Fig. 4) into live (Annexin V and 7-AAD
negative, region 1 lower left of FACS plot), apoptotic
(Annexin V positive, 7-AAD negative, region 2 center), or
necrotic (Annexin V positive, 7-AAD positive, region 3 upper
right). Typical data from two individual mice, one infected for 6
wk, one for 38 wk, are shown. The left-hand panel shows
apoptosis among total splenocytes, increasing from 11% at 6 wk to 15%
at 38 wk. The central panel shows splenocytes clearly
staining with anti-CD4 mAb 6 wk postinfection, but by 38 wk these
cells were markedly depleted, either by loss of the cells themselves,
or down-regulation of the marker. When analysis of apoptotic cells was
gated on the region containing CD4+ cells, the
percentages of apoptotic cells were 30% at 6 wk and 50% at 38 wk,
indicating that most of the apoptosis occurred among T cells.
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) show graphically the decline in
percentage of CD4+ and CD8+
cells over time of infection, and the increase in apoptotic
CD4+ and CD8+ cells. The
percentage of CD4+ cells declined to 1/3, and
CD8+ cells to less than half normal by 38 wk.
Conversely there were 4–5 times normal numbers of apoptotic
CD4+ and CD8+ cells in the
38-wk infected spleens. Apoptotic cells among the
CD4-CD8- population
increased 2-fold at the same time, but remained only 13% of that
population, compared with almost 50% of CD4+ and
35% of CD8+ cells. Apoptosis was thus highly
selective for T lymphocytes.
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We wished to examine the relationship between immunological
changes over the course of infection in individual mice. Technical
limitations to the handling of large numbers of specimens precluded the
possibility of examining all parameters at all time points in one
experiment. However, observations were remarkably consistent from one
experiment to the next. Therefore, data from all experiments in which
bacterial counts, apoptosis, IFN- production, and mouse weight were
all documented on individual mice are pooled in Table III.
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production was still high. This points to an
inadequacy of control of infection, even in the presence of IFN- to
activate macrophages. Nevertheless, two mice at 36 wk (mice 19 and 20)
that produced more IFN- than their cohorts were also healthier,
having lower bacterial numbers and heavier body weight. Even these mice
produced suboptimal IFN-, and indeed all mice infected for 34 wk or
more were showing a marked deficiency in IFN-. The decline in body
weight began at this time for some mice (underlined), but many were
still relatively robust after 37 wk. Therefore, weight loss could be
said to occur only in the final stages of infection, as indeed was
apparent in Fig. 1.
The relationship between apoptosis and IFN- production was of prime
interest. The greatest increase in apoptosis was between uninfected
mice and the earliest observations made at 6 wk (Fig. 5). However,
apoptosis of CD4+ cells steadily increased over
the succeeding months (Fig. 5, Table III). When apoptosis and IFN-
production were related in individual mice, there was generally a
strong correlation between apoptosis and declining IFN- (Fig. 6). Three mice, however, showed low
IFN- production despite only moderate apoptosis, and four showed
high IFN- production despite moderately high rates of apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and depletion of CD4 T cells or IFN- by mAb
exacerbated infection (15). The present study extends this
time course to more than 35 wk, during which bacterial numbers and
apoptosis of T cells steadily increased. Loss of IFN- production did
not occur until bacterial numbers had increased to
>108.5 in the lungs of the mice and apoptosis
was affecting 30% or more of their CD4+ T cells.
This was soon followed by acute weight loss, and the mice would
undoubtedly have died if they were not killed for experimental or
humane reasons.
The abrupt loss of immune reactivity measured by IFN- production
observed in the M. avium-infected mice provides an
extraordinarily useful model. There exists an earlier description of
loss of delayed-type hypersensitivity in Mycobacterium
tuberculosis-infected mice, which was followed by an increase in
bacterial numbers and death of the mice (23). This
occurred apparently at random, beginning 12 wk after intranasal
infection with the virulent H37Rv strain of M. tuberculosis.
Some mice survived 40 wk or more without loss of delayed-type
hypersensitivity. These studies, however, predated our understanding of
the role of T lymphocytes in such responses.
In the present experiments, IFN- production by T lymphocytes in the
M. avium-infected mice was measured using three different
approaches: bioassay of cytokine released after 72 h into the
supernatant of Ag-stimulated splenocyte culture; ELISPOT assay of the
numbers of IFN--producing cells in similar cultures and by staining
for intracytoplasmic cytokine after 6 h of in vitro restimulation
of T cells with anti-CD3 mAb. Although there were obvious
differences in sensitivity of the methods of assay, the conclusions
were the same. Analysis by all three methods of IFN- production over
the course of infection showed a sustained production up to 28 wk
postinfection, followed by loss of IFN- production by all mice at
34 wk.
Analysis of cytokine production by ICC staining showed that at their
peak as many as 10% of CD4+ and of
CD8+ T cells produced IFN- after in vitro
restimulation with anti-CD3 Ab. Because of the lower percentage of
CD8+ T cells in the spleen, their absolute
contribution to IFN- production was less than that of
CD4+ T cells. Nevertheless, the fact that so many
CD8+ T cells produced IFN- was remarkable,
because it has not been clear that they contribute to resistance
against this organism (15). In addition, there were
clearly cells that expressed neither CD4 nor CD8 markers, but that
produced IFN- under the conditions used. Because stimulation with
anti-CD3 was necessary to demonstrate IFN- production, it
suggests that the TCR is present on these cells, and that they may be
T cells or perhaps ß T cells in which CD4 or CD8 has been
down-regulated. Their identity is under investigation. It should be
noted that restimulation with anti-CD3 could include bystander
cells, and indeed the estimate of numbers of cells responding to
specific M. avium Ag using the ELISPOT assay was lower.
Nevertheless, in a viral infection, a similarly massive increase in the
numbers of IFN--secreting cells has been found not to represent
bystanders, but Ag-specific cells (24, 25).
In the final phases of infection, with the decline in IFN-
production, there was no indication of a switch to Th2 cytokine
profile. We were unable to demonstrate IL-4 production in the
supernatant of bulk cultures, nor was there IL-4 demonstrable in
the cytoplasm of anti-CD3-stimulated cells (results not shown). A
positive control comprising a transformed cell line producing IL-4
(26) was successfully used to validate the cytoplasmic
staining assay. On the other hand, the loss of IFN- production
reflected in part the loss of CD4+ and
CD8+ T cells. The percentage of
CD4+ T cells producing IFN- declined to
one-third normal levels by 30 or more weeks, while the number of
CD4+ T cells themselves fell from 25% in normal
uninfected mice to <10% in the final phase of infection. Taken
together this meant that the absolute number of IFN--producing
CD4+ T cells measured by ICC staining fell from
12 x 105 to 1 x
105 per spleen.
The other striking finding was the increase in apoptosis during the
course of infection. Apoptosis was highly selective, affecting up to
50% of CD4+ T cells and 30% of
CD8+ T cells, but only 12% of total spleen
cells. A number of studies have implicated apoptosis in chronic
viral infection, including HIV (27) and experimental
infection with lymphocytic choriomeningitis virus (12). It
has been suggested (12) that full induction of immune T
cells involves expansion and eventual death of the effector T cell
within a relatively short time. When only a few precursor T cells are
stimulated in a staggered fashion, the population as a whole persists
and is restimulated at low levels by persisting Ag to form a pool of
memory T cells. However, there is evidence in the lymphocytic
choriomeningitis virus-infected mice that in the presence of high
levels of persisting Ag, virtually all T cells are driven to terminal
differentiation, apoptosis, and clonal abortion. Thus, the final stages
of such an infection represent a state of immunological tolerance. It
is possible this is occurring in the terminal stages of M.
avium infection when the mice have been exposed to extraordinarily
high (up to 108 or 109)
numbers of bacteria for 8–9 mo. The abrupt loss of IFN- recall is
certainly indicative of tolerance. However, further work is required to
demonstrate the antigenic specificity that is a hallmark of
tolerance.
A remarkable feature of the end phase of M. avium infection
is the fact that all of the mice reached the same stage over a very
short period of time, perhaps the last 5 wk of a 33–38-wk course of
infection. It is difficult to prove what is cause and what effect. We
therefore investigated the relationship between IFN-, apoptosis,
bacterial numbers, and weight loss in individual mice over time. Which
came first? Did the decline of IFN- production allow the increase in
bacterial numbers or was it the result? Was increasing apoptosis
responsible for the loss of IFN- production? Was weight loss the
final expression of decline?
In fact, there was a gradual increase in bacterial numbers even before
loss of IFN- production. Thus, at 28 wk, there was a 10-fold
increase in bacterial numbers in the lungs compared with 13 wk, even
though IFN- was produced to high titers by cultured lymphocytes.
This indicates an underlying inadequacy of control of the infection,
despite the presence of IFN- to activate the macrophages. We know
that IFN- is essential for the control of early infection, because
depletion by injection of mAb to IFN- itself (15) or to
IL-12, which controls secretion of IFN- (16),
exacerbates infection. In the present experiments, IFN- production
underwent dramatic reduction between 28 and 34 wk. Given the steady
march of bacterial numbers before and after this event, loss of IFN-
production appears to be the result, rather than the cause, of high
bacterial numbers. Interestingly, two mice that were still producing
appreciable, although reduced, amounts of IFN- at 36 wk
postinfection were healthier than their cohorts, having fewer bacteria
and heavier body weight. For some mice, weight loss began at the same
time as IFN- production ceased, while other mice maintained
their weight for a longer time. Some still appeared relatively healthy
at 37 wk, so weight loss seems indeed to be a sign of terminal stages
of infection.
In assessing the relationship between apoptosis and IFN- recall in
individual mice, there was generally a striking correlation between low
IFN- production and high apoptotic rates. Four mice in which more
than 25% of CD4+ T cells were apoptotic still
produced high titers of IFN-, which would be compatible with
apoptosis preceding and perhaps causing the decline in IFN-
production. However, 3 of the 30 mice produced low titers of IFN-
despite only moderate levels of apoptosis, suggesting the parallel
between apoptosis and IFN- decline may be coincidental rather than
causal. Thus, the question of whether the terminal differentiation and
apoptosis of T lymphocytes are responsible for the final decline of
these mice must for the moment remain unanswered.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Cheers, Department of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia. E-mail address:
3 Abbreviations used in this paper: ELISPOT, enzyme-linked immunospot. ICC, intracellular cytokine; 7-AAD, 7-amino actinomycin D.
Received for publication February 12, 1999. Accepted for publication June 3, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. G. Feng, C. M. Collazo-Custodio, M. Eckhaus, S. Hieny, Y. Belkaid, K. Elkins, D. Jankovic, G. A. Taylor, and A. Sher Mice Deficient in LRG-47 Display Increased Susceptibility to Mycobacterial Infection Associated with the Induction of Lymphopenia J. Immunol., January 15, 2004; 172(2): 1163 - 1168. [Abstract] [Full Text] [PDF]
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B. Gilbertson and C. Cheers Generalized Immunological Decline during Long-Term Experimental Infection with Mycobacterium avium Infect. Immun., December 1, 2002; 70(12): 7145 - 7148. [Abstract] [Full Text] [PDF]
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S. I. Mannering and C. Cheers Interleukin-2 and Loss of Immunity in Experimental Mycobacterium avium Infection Infect. Immun., January 1, 2002; 70(1): 27 - 35. [Abstract] [Full Text] [PDF]
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V. Nagabhushanam and C. Cheers Non-Major Histocompatibility Complex Control of Antibody Isotype and Th1 versus Th2 Cytokines during Experimental Infection of Mice with Mycobacterium avium Infect. Immun., March 1, 2001; 69(3): 1708 - 1713. [Abstract] [Full Text] [PDF]
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, Infected mice; 
, uninfected
age-matched mice.
