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Infect Immun, August 1998, p. 3562-3568, Vol. 66, No. 8
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Altered Cytokine Production and Impaired
Antimycobacterial Immunity in Protein-Malnourished Guinea
Pigs
Guixiang
Dai and
David N.
McMurray*
Department of Medical Microbiology and
Immunology, College of Medicine, Texas A&M University Health
Science Center, College Station, Texas 77843
Received 17 December 1997/Returned for modification 18 February
1998/Accepted 13 May 1998
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ABSTRACT |
Protein malnutrition leads to multiple detrimental alterations of
host immune responses to mycobacterial infection. In this study, we
demonstrated that splenocytes from low-protein (LP) guinea pigs
vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal
macrophages, despite the fact that they were able to control the
accumulation of virulent tubercle bacilli in cocultured syngeneic
peritoneal macrophages from normally nourished guinea pigs as
successfully as did those from high-protein (HP) counterparts.
Vaccine-induced growth control of virulent M. tuberculosis
H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes
but not CD8 cells. Tuberculin (purified protein derivative
[PPD])-induced lymphoproliferation was markedly impaired in
vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes
significantly decreased lymphocyte proliferation whereas CD8 cell
depletion did not. Protein malnutrition also impaired the abilities of
cells from vaccinated LP guinea pigs to produce cytokines, including
interferon, tumor necrosis factor alpha (TNF-
) and transforming
growth factor beta (TGF-
), in response to PPD, despite the
demonstration of higher serum levels of TNF-
and TGF-
after an
intravenous injection of PPD into LP guinea pigs. In contrast,
peritoneal macrophages from protein-malnourished guinea pigs produced a
higher level of TGF-
4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls.
These results suggest that dietary protein malnutrition impairs
vaccine-induced resistance to M. tuberculosis, in part, by
altering the cytokine profile to favor macrophage deactivation.
 |
INTRODUCTION |
Tuberculosis causes approximately
1.5 billion latent infections, 8 million new clinical cases, and 3 million deaths annually, making it the most prevalent infectious
disease in the world (9). The emergence of
multidrug-resistant tuberculosis and the pandemic of AIDS have further
exacerbated the situation (1, 11). The host protective
immune mechanisms against infection with Mycobacterium tuberculosis depend critically on the interactions and cooperation between monocytes-macrophages, T lymphocytes, and their cytokines (22), which are sensitive to nutritional insult (5-8,
18).
The incidence of tuberculosis is unusually high among malnourished
people, including the elderly, the homeless, alcoholics, drug abusers,
and human immunodeficiency virus-infected individuals (43).
The reactivation of latent or previously subclinical tuberculous infections may be related, in part, to deteriorating nutritional status
(42). Protein malnutrition has been identified as an important risk factor for the predisposition to intracellular infections leading to death (47). There is a strong
association between protein malnutrition and impairment of a range of
immune functions, principally those mediated by T lymphocytes, which are known to be essential for resistance to tuberculosis (18, 23,
28). Studies of experimental animals indicate that protein deprivation significantly impairs macrophage activation and granuloma development (38) and alters T-cell functions and cytokine
production (5-8, 18). Previous reports from this laboratory
have demonstrated that chronic, moderate protein deficiency severely
impairs T-cell functions and protective efficacy of M. bovis
BCG vaccination (32-34). Recently, it was reported that
protein calorie malnutrition markedly enhanced bacterial growth and
dissemination in mice infected with the attenuated BCG vaccine strain
(5) and diminished the expression of gamma interferon
(IFN-
) and tumor necrosis factor alpha (TNF-
) in the lungs of
mice infected by the intravenous route with a very high dose of the
virulent M. tuberculosis Erdman strain, resulting in rapidly
fatal tuberculosis infection and a markedly elevated bacillary load in
the lungs of protein-deficient mice (6). The precise
mechanisms by which protein malnutrition impairs host antimycobacterial
immunity remain largely unknown.
In this study, we investigated the effects of protein malnutrition on
the interaction between immune lymphocytes and M. tuberculosis H37Rv-infected macrophages, the roles of CD4 and CD8
T-cell subpopulations, and the production of cytokines in M. tuberculosis H37Ra-vaccinated guinea pigs.
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MATERIALS AND METHODS |
Experimental animals.
Both pathogen-free inbred strain 2 guinea pigs (NCI-Frederick Cancer Research Facility, Frederick, Md.)
and outbred Hartley strain guinea pigs (Charles River Breeding
Laboratories, Inc., Wilmington, Mass.), weighing 200 to 300 g,
were used in this study. The animals were individually housed in an
air-filtered environment, within polycarbonate cages with stainless
steel grid floors and feeders. They were given food and tap water ad
libitum. Each animal was randomly assigned to an experimental diet.
Experimental diets.
Purified experimental diets were
obtained from a commercial supplier (Dyets, Inc., Bethlehem, Pa.) and
formulated to our specifications by inversely varying the proportions
of cornstarch and ovalbumin to obtain the desired protein content. The
exact diet composition was published previously (30). The
low-protein (LP) diet contained 10% ovalbumin, and the high-protein
(HP) diet contained 30% ovalbumin. The diets were isocaloric and
identical in every nutrient except protein. The third diet used was a
commercial guinea pig chow (Ralston Purina, St. Louis, Mo.).
M. tuberculosis H37Ra vaccination.
Two weeks
after the experimental diets were started, guinea pigs were infected
with viable, attenuated M. tuberculosis H37Ra (ATCC 25177;
American Type Culture Collection, Rockville, Md.). Each animal received
0.1 ml of sterile physiological saline containing about 2 × 103 viable bacilli subcutaneously in the left inguinal
area. The viability of the inoculum was determined by plating
appropriate dilutions on Middlebrook 7H10 agar (Difco Laboratories,
Detroit, Mich.).
Necropsy procedure.
Three weeks after M. tuberculosis H37Ra vaccination, all guinea pigs were treated daily
with streptomycin (40 mg/kg of body weight) and rifampin (20 mg/kg) for
3 weeks to sterilize the animals, as determined by plating lung and
spleen tissue dilutions on M7H10 agar and observing no bacillary
growth. Some guinea pigs were injected via the ear vein with purified
protein derivative (PPD; 1 mg/kg) 2 h before euthanasia.
Euthanasia was carried out by the intramuscular injection of 2 ml of
sodium pentobarbital (Fort Dodge Laboratories, Inc., Fort Dodge, Iowa).
A blood sample was obtained immediately via cardiac puncture. Sera were
separated by centrifugation and stored at
70°C until use. The
abdominal cavity of each guinea pig was opened aseptically, and the
spleen was removed for lymphocyte isolation by gentle homogenization with a glass homogenizer. The homogenates were treated with ACK lysing
buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM
Na2EDTA [pH 7.2 to 7.4]) for 3 to 5 min at room
temperature to destroy erythrocytes, the cells were washed three times
in RPMI 1640 medium, viability was determined by trypan blue exclusion,
and the number of cells was adjusted to the desired concentration in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 100 U of penicillin per ml, 100 µg of streptomycin per
ml, and 2 µM L-glutamine (working medium).
Isolation of peritoneal macrophages.
Guinea pigs were
injected intraperitoneally with 5 ml of 3% thioglycolate and
euthanized 4 to 5 days later by intramuscular injection of 2 ml of
sodium pentobarbital. The peritoneal cavity was washed with 20 ml of
phosphate-buffered saline (PBS) containing 20 U of heparin per ml and
2% FBS. Peritoneal cells were collected, washed three times with and
suspended in RPMI 1640 working medium, and incubated in plastic petri
dishes at 37°C for 1 to 2 h. The dishes were then placed at
4°C for 20 min, and the adherent cells were scraped off, collected,
washed twice in working medium, counted by the trypan blue exclusion
method, and adjusted to the desired concentration in working medium
without streptomycin.
Depletion of CD4 T cells, CD8 T cells, or both.
For
depletion of T-lymphocyte subpopulations, the following monoclonal
antibodies against the specified guinea pig T-cell markers were used:
CT7, anti-CD4 (35, 44); CT6, anti-CD8 (35, 44);
and CT5, anti-pan-T-cell marker (4, 44). All were purchased
from Serotec Inc., Partners 1, Raleigh, N.C. Different T-cell
subpopulations were depleted by treatment of 5 × 106
to 1 × 107 cells with a 1:500 dilution of anti-CD4,
anti-CD8, or anti-pan-T-cell marker monoclonal antibody and a 1:8
dilution of normal guinea pig serum in RPMI 1640 working medium.
Lymphocyte suspensions were incubated with monoclonal antibodies for 45 min at 4°C, washed twice with 3 to 5 ml of cold PBS, and resuspended
in working medium containing diluted guinea pig serum. After incubation
for 30 min at 37°C, cells were washed twice and incubated with
diluted serum for another 30 min at 37°C. After incubation, cells
were washed three times, assessed for viability by trypan blue
exclusion, and adjusted to 2 × 106 cells/ml in
working medium without streptomycin. Pan-T cells, CD4 T cells, and CD8
T cells in the suspensions accounted, on average, for 34.1, 26.9, and
13.6%, respectively, before depletion and 5.4, 6.2, and 3.4%,
respectively, after depletion, as determined by flow cytofluorometric
analysis (26). Therefore, these depletion procedures, on
average, reduced T cells by 84%, CD4 cells by 77%, and CD8 cells by
75%.
Coculture of infected macrophages and lymphocytes.
Adherent
peritoneal macrophages were cultured with live, dispersed virulent
M. tuberculosis H37Rv organisms (ATCC 27294) at a ratio of
approximately 100 macrophages per mycobacterium at 37°C for 2 to
4 h. Then differently treated lymphocytes were added, and the
cells were cultured for another 7 days. The cultures were lysed with
distilled water and briefly sonicated for 3 to 5 s to disperse the
mycobacteria, using an Ultrasonics sonicator (Heat System-Ultrasonics,
Inc., Plainview, N.Y.) with an output control setting of 3.0. The
lysates were diluted in physiological saline, vigorously vortexed, and
plated onto M7H10 agar plates. After incubation at 37°C for 3 weeks,
the colonies were counted and expressed as CFU of mycobacteria per
culture.
Preparation of lymphocyte and peritoneal macrophage culture
supernatants.
Peritoneal macrophages at 106 cells/ml
in RPMI 1640 containing 15 µg of PPD (Statens Seruminstitut,
Copenhagen, Denmark) per ml were cultured in 24-well culture plates at
1 ml per well. After incubation at 37°C in 5% CO2 for 24 or 48 h, cell-free supernatants were collected after
centrifugation of the cultures at 1,500 rpm for 10 min and stored at
70°C until use. For M. tuberculosis H37Rv-induced
production of TNF-
and transforming growth factor
1 (TGF-
1),
0.25 ml of peritoneal macrophages at 4 × 106 cells/ml
was well mixed with 0.25 ml of live, dispersed M. tuberculosis H37Rv organisms at a multiplicity of infection of 1:1
and cultured at 37°C for 4 h, and then 0.5 ml of splenocytes at
2 × 105 cells/ml was added. After incubation for
another 2 or 4 days, cell-free supernatants were collected after
centrifugation of the cultures at 1,500 rpm for 10 min and stored at
70°C until use.
To assess the effect of diet on IFN production, primed splenocytes at
2 × 106 cells per ml in RPMI 1640 containing 15 µg
of PPD per ml were cultured in 24-well culture plates in a total volume
of 1 ml per well. After incubation at 37°C in 5% CO2 for
48 h, cell-free supernatants were collected after centrifugation
of the cultures at 1,500 rpm for 10 min and stored at
70°C until
use.
TGF-
bioassay.
The Mv-1-Lu mink lung cell line (ATCC
CCL-64) was grown in Eagle's minimal essential medium containing 10%
FBS and used to measure TGF-
bioactivity as described previously
(37). Briefly, triplicate cultures were set up by seeding
2 × 104 Mv-1-Lu cells per well in 100 µl of medium
in 96-well plates and adding 100 µl of diluted samples, followed by
incubation for 24 h at 37°C in a 5% CO2 humidified
incubator. All samples were treated with 0.12 N HCl for 15 min at room
temperature and then neutralized with 0.1 M HEPES-0.144 N NaOH before
use. The cell cultures were labeled with 1 µCi of
[3H]thymidine (6.7 Ci/mmol; ICN Radiochemicals, Irvine,
Calif.) per well for the final 8 h and harvested onto fiberglass
filter disks, and the uptake of [3H]thymidine was
measured in a liquid scintillation counter (LS8000; Beckman
Instruments, Fullerton, Calif.). A standard curve was generated with
recombinant human TGF-
1 (R&D Systems, Minneapolis, Minn.) over a
concentration range of 0.008 to 4 ng/ml. This assay was sensitive to
TGF-
activity of 40 pg/ml.
TNF-
bioassay.
Macrophage culture supernatants were
assessed for TNF-
activity by measuring their cytotoxicity on L929
cells as described by Espevik et al. (12). Briefly, 0.1-ml
aliquots of L929 cell suspension (4 × 105 cells/ml)
were pipetted into 96-well culture plates and incubated overnight. The
following day, 0.1 ml of dilutions of culture supernatants or various
concentrations of recombinant human TNF-
(rhTNF-
; R&D Systems)
and actinomycin D at a final concentration of 2 µg/ml were added to
each well. After incubation for 18 h, 10 µl of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] at a
concentration of 5 mg/ml in PBS was added, and the cells were further
cultured for 4 h. After the supernatants were decanted, 0.1 ml of
isopropanol with 0.04 N HCl was added to each well, and the plates were
incubated for 3 h. The plates were read by an enzyme-linked
immunosorbent assay (ELISA) reader (Dynatech MR 5000; Dynatech
Laboratories, Chantilly, Va.), using a test wavelength of 570 nm and a
reference wavelength of 630 nm. A standard curve was generated with
rhTNF-
. To verify that the assay was detecting TNF-
-induced
cytotoxicity, some of the supernatant samples were incubated with
rabbit anti-rhTNF-
neutralizing antibody (R&D Systems) and some with
normal rabbit immunoglobulin G at 37°C for 2 h prior to the
addition of the supernatants to the target cell cultures. This assay
was sensitive to TNF-
activity of 25 pg/ml.
IFN bioassay.
Total IFN bioactivity in the culture
supernatants was measured by inhibition of the cytopathic effect of
vesicular stomatitis virus (VSV) on mouse L929 cells (19).
Briefly, serial twofold dilutions of 50 µl of IFN-containing
supernatants were made in Iscove's modified Dulbecco medium
(GIBCO BRL, Grand Island, N.Y.) without serum in a 96-well
plate, and 0.1 ml of L929 cells (5 × 104 cells) was
added per well. Following overnight incubation, cells were challenged
with 50 µl of VSV at a multiplicity of infection of 1. After
incubation for 24 h, the supernatant from each well was removed,
and the cells were washed and fixed with 0.1 ml of 5% formalin at room
temperature for 10 min. Then the cells were washed and stained with 0.1 ml of crystal violet solution for 10 min. Finally, the plate was rinsed
and dried. After addition of 0.1 ml of 100% methanol to each well, the
plates were read at 595 nm on a microtiter ELISA plate reader. A
standard curve was generated with recombinant human IFN-
(R&D
Systems). This assay was sensitive to IFN-
activity of 20 U/ml but
detects all three species of IFN.
Lymphocyte blastogenesis.
Splenocytes were distributed into
96-well flat-bottom tissue culture plates (Corning Glass Works,
Corning, N.Y.) at 2 × 105 cells per well in RPMI 1640 working medium. Triplicate cultures were stimulated with PPD at a final
concentration of 15 µg/ml. For some experiments, 10 µg of
anti-human interleukin-2 (IL-2) antibody (Genzyme Corporation,
Cambridge, Mass.) per ml was added to the cultures. The cultures were
incubated for 3 days at 37°C in a 5% CO2 atmosphere,
labeled with 1.0 µCi of tritiated thymidine per well for another
18 h, and harvested onto glass wool fiber filters, using a cell
harvester. [3H]thymidine incorporation into cellular DNA
was measured in a liquid scintillation counter. The results were
expressed as mean net counts per minute (counts per minute in
stimulated wells minus counts per minute in control wells).
Statistical analysis.
The analysis of variance was used to
test for the effect of dietary treatment on the dependent variables
measured. When appropriate, Student's t test was used to
analyze the significance of differences between group means. A 95%
confidence level was set for all tests.
 |
RESULTS |
Influence of dietary protein malnutrition on the growth containment
of M. tuberculosis H37Rv induced in peritoneal macrophages
by M. tuberculosis H37Ra-primed T lymphocytes.
The
initial experiments showed that cocultures of peritoneal macrophages
with syngeneic splenocytes from inbred strain 2 guinea pigs vaccinated
6 weeks previously with viable, attenuated M. tuberculosis
H37Ra inhibited the growth of virulent tubercle bacilli in a
dose-dependent fashion (Fig. 1). In
cocultures with syngeneic peritoneal macrophages derived from normally
nourished donor guinea pigs, splenocytes from LP guinea pigs vaccinated
6 weeks previously with M. tuberculosis H37Ra were able to
control the accumulation of virulent tubercle bacilli in cultured
macrophages as successfully as those from HP guinea pigs (Fig.
2). Only the CD4 T-cell population appeared to be necessary in both diet groups to mediate
antituberculosis resistance, as evidenced by the abrogation of this
vaccine-induced bacillary control by the depletion of CD4 T
lymphocytes, or CD4 plus CD8 T cells, but not by the depletion of CD8
cells alone (Fig. 2).

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FIG. 1.
Growth containment of virulent M. tuberculosis H37Rv organisms induced in peritoneal macrophages by
M. tuberculosis H37Ra-primed lymphocytes. Peritoneal
macrophages were infected with live, dispersed virulent M. tuberculosis H37Rv at 37°C for 2 to 4 h and then cocultured
for 7 days with different numbers of syngeneic splenocytes derived from
M. tuberculosis H37Ra-vaccinated and nonvaccinated inbred
strain 2 guinea pigs. The cultures were harvested and streaked onto
M7H10 agar plates. After 3 weeks of incubation, the CFU were counted.
The means ± standard errors of the means for four animals in each
group are shown.
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FIG. 2.
Influence of dietary protein malnutrition on the
interactions between lymphocytes and macrophages. Peritoneal
macrophages from normally nourished donors were infected with live,
dispersed virulent M. tuberculosis H37Rv at 37°C for 2 to
4 h and then cocultured for 7 days with whole syngeneic
splenocytes or splenocytes depleted of CD4 T cells, CD8 T cells, or
both. The splenocytes were derived from inbred strain 2 guinea pigs
vaccinated with M. tuberculosis H37Ra and maintained on the
LP or HP diet. The cultures were harvested and streaked onto M7H10 agar
plates. After 3 weeks of incubation, the CFU were counted. The
means ± standard errors of the means for four animals in each
group are shown.
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Despite the fact that splenocytes from vaccinated LP guinea pigs were
able to control the growth of virulent tubercle bacilli
as successfully
as cells from HP guinea pigs did in coculture
with syngeneic peritoneal
macrophages derived from normally nourished
guinea pigs (Fig.
2), they
failed to do so in coculture with autologous
(i.e., protein-deprived)
peritoneal macrophages, as indicated
by a significant increase in the
number of tubercle bacilli recovered
from LP cultures (HP = 11,420 ± 3,200 and LP = 28,200 ± 3,600
CFU per
culture;
P < 0.05). These results indicate that
protein
malnutrition impairs the macrophage-lymphocyte interaction
and/or
macrophage antimicrobial effector functions.
PPD-driven lymphocyte blastogenesis.
Figure
3 illustrates the impact of protein
malnutrition on the proliferative response of splenocytes 6 weeks
following M. tuberculosis H37Ra vaccination. Lymphocytes
derived from LP guinea pigs had a markedly depressed proliferative
response to PPD compared with that from HP guinea pigs (P < 0.01). Lymphocyte subpopulation depletions revealed that CD4 T-cell
depletion significantly reduced the PPD-induced lymphoproliferation in
HP guinea pigs (P < 0.05), while CD8 T-cell depletion
did not. In contrast, neither CD4 nor CD8 T-cell depletion
significantly altered the suppressed PPD-induced responses of
splenocytes from LP guinea pigs. These results suggest that protein
malnutrition alters the development and/or proliferative function of
antigen-specific lymphocytes.

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FIG. 3.
Dietary protein malnutrition depresses the proliferative
responses of lymphocyte subpopulations in guinea pigs. Inbred strain 2 guinea pigs maintained on the LP or HP diet were vaccinated with
M. tuberculosis H37Ra. Six weeks later, guinea pigs were
euthanized and splenocytes were prepared. After no depletion or
depletion of CD4 T cells, CD8 T cells, or both, the splenocytes were
stimulated with PPD at 15 µg/ml for 3 days and labeled with
[3H]thymidine for another 18 h. The cultures were
harvested, and proliferation was measured as net thymidine uptake. The
means ± standard errors of the means for eight guinea pigs per
treatment are shown.
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Impact of protein malnutrition on the production of cytokines.
The effect of dietary protein malnutrition on TNF-
production was
evaluated by measuring TNF-
bioactivity in the sera of M. tuberculosis H37Ra-vaccinated guinea pigs with or without
intravenous injection of PPD and in the culture supernatants of
peritoneal macrophages stimulated with PPD or infected with M. tuberculosis H37Rv (Table 1). Six
weeks following vaccination, sera were separated by centrifugation of
blood samples and measured for TNF-
bioactivity. Protein
malnutrition resulted in a significantly higher serum level of
bioactive TNF-
in guinea pigs vaccinated with M. tuberculosis H37Ra. An intravenous injection of PPD boosted
remarkably the serum level of bioactive TNF-
in both LP and HP
guinea pigs, with the level of TNF-
in sera from LP guinea pigs
rising significantly higher than that from HP guinea pigs (P < 0.001). In contrast, peritoneal macrophages derived from HP guinea pigs
produced a much higher amount of TNF-
than did those from LP guinea
pigs when stimulated in vitro with PPD (P < 0.01) or infected
with M. tuberculosis H37Rv for 2 and 4 days (P < 0.001). These results indicate that protein malnutrition impairs the
capacity of macrophages to produce TNF-
in response to mycobacteria
and their antigens, at least in vitro, and that elevated serum TNF
bioactivity in LP animals may not be due to increased production by
macrophages.
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TABLE 1.
Effect of dietary protein deprivation on the production
of TNF- in vivo and in vitro in guinea pigs vaccinated with
M. tuberculosis H37Ra
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Figure
4 illustrates that vaccinated LP
guinea pigs had a significantly higher level of TGF-

in their serum
than HP guinea
pigs after an intravenous injection of PPD (P < 0.05), while peritoneal
macrophages from LP guinea pigs produced
significantly less TGF-
in response to PPD in vitro than did those
from HP guinea pigs
(P < 0.01). In contrast, as seen in Fig.
5, peritoneal macrophages
from immune LP
guinea pigs produced a significantly higher level
of TGF-

when
infected with
M. tuberculosis H37Rv for 4 days than
did
those from HP guinea pigs (P < 0.01).

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FIG. 4.
Dietary protein malnutrition alters TGF- production
in guinea pigs vaccinated with M. tuberculosis H37Ra.
Outbred Hartley strain guinea pigs were vaccinated with M. tuberculosis H37Ra and maintained on the LP or HP diet for 6 weeks. Sera were collected following an intravenous injection of 1 mg
of PPD/kg of body weight 2 h prior to euthanization. Peritoneal
macrophages were stimulated with PPD at 15 µg/ml for 48 h.
Cell-free supernatants were collected and measured for TGF-
bioactivity. Data for six guinea pigs per treatment are expressed as
mean ± standard error of the mean.
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FIG. 5.
Dietary protein malnutrition enhances the production of
TGF- by peritoneal macrophages infected with M. tuberculosis H37Rv in guinea pigs. Peritoneal macrophages derived
from outbred Hartley strain guinea pigs vaccinated with M. tuberculosis H37Ra and maintained on the LP or HP diet were
infected with live, dispersed M. tuberculosis H37Rv for 2 to
4 h and then cultured for 2 and 4 days. Cell-free supernatants
were collected and measured for TGF- bioactivity. Data for six
guinea pigs per treatment are expressed as mean ± standard error
of the mean.
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The production of bioactive IFN by splenocytes from vaccinated guinea
pigs in response to PPD was suppressed by protein malnutrition.
Immune
splenocytes from HP guinea pigs produced a significantly
higher amount
of bioactive IFN than did those from LP guinea pigs
(HP = 326.4 ± 37 and LP = 194.8 ± 24.5 U/ml;
P < 0.01) following
culture for 48 h with 15 µg
of PPD per ml.
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DISCUSSION |
The detrimental effects of nutritional deficiencies on
tuberculosis could result from alterations in the individual protective functions of, or the interaction between, T lymphocytes and
macrophages, which are the major cell types mediating antimycobacterial
immunity (15, 22, 39). In the present study, we demonstrated
that splenocytes derived from inbred strain 2 protein-malnourished guinea pigs vaccinated previously with M. tuberculosis H37Ra
were able to cooperate with syngeneic macrophages from normally
nourished donors to control the accumulation of virulent tubercle
bacilli inside infected macrophages (Figure 2) but not with autologous macrophages from the same protein-deficient environment. The fact that
lymphocytes from LP guinea pigs could activate macrophages from
normally nourished guinea pigs suggests that protein deficiency did not
alter this activity, at least in vitro. On the other hand, the
inability of LP lymphocytes to activate LP macrophages provides evidence that protein malnutrition impairs the productive interaction between macrophages and T lymphocytes and/or the acquisition of mycobactericidal-mycobacteriostatic activity by LP macrophages in the
presence of adequate activation signals.
Adoptive transfer experiments conducted previously in this laboratory
demonstrated that immune lymphocytes derived from protein-deficient guinea pigs have a relatively low ability to protect normally nourished
recipients from respiratory challenge with virulent M. tuberculosis H37Rv, indicating that protein deficiency
prevented guinea pigs from generating a population of antigen-specific
immune lymphocytes and/or impaired the proliferative capacity of these cells (27). In this study, we demonstrated that immune
lymphocytes derived from protein-deficient guinea pigs responded poorly
to PPD (Fig. 3) and were unable to control the accumulation of virulent tubercle bacilli in the cocultures with autologous macrophages. These
findings could be interpreted to result from a defective generation of
antigen-specific immune lymphocytes caused by protein deficiency
because little evidence for macrophage defects in protein-deficient guinea pigs was found in previous studies (24, 31, 33). This
interpretation is compatible with the results of adoptive transfer
experiments described above (27). Further experiments will
be carried out to verify if there is a defective generation of
antigen-specific immune lymphocytes in LP guinea pigs or if the defect
is in cell-to-cell communication. The observation that immune
lymphocytes from protein-deficient guinea pigs were able to activate
macrophages from normally nourished guinea pigs to control the growth
of virulent mycobacteria in vitro could be explained by the relatively
high number of immune lymphocytes concentrated in the cocultures and
the closeness of contact between lymphocytes and macrophages, which may
facilitate the generation of adequate activation signals for
macrophages and thus compensate for the functional alteration of these
cells.
Both CD4 and CD8 T cells have been shown to be required in the control
of mycobacterial infection in mice (14, 25, 36). However,
the precise role of each of these cell types has not been fully
elucidated, and no data on the role of T-cell subsets in the guinea pig
have been published. In our in vitro macrophage coculture system, CD4 T
cells were demonstrated to be the major effector cells in containing
the growth of virulent M. tuberculosis H37Rv, while the
depletion of CD8 T cells did not alter the outcome (Figure 2). This
result suggests that CD8 T cells contribute little, if any, to the
growth control of virulent tubercle bacilli either by mediating target
cell lysis or by cytokine production in this coculture system.
Immunization with M. tuberculosis H37Ra, which is attenuated
and thought to reside exclusively in the phagosome (39), may
not effectively activate major histocompatibility complex class
I-restricted CD8 T cells. Therefore, one reasonable explanation for our
result is that very few, if any, antigen-reactive CD8 T cells are
present in the cocultures.
Cytokines play a central role in mediating antimycobacterial immunity.
IL-2 is required to initiate and amplify immune responses. IFN-
and
TNF-
are important macrophage-activating cytokines and crucial in
the immune response to tuberculosis (10, 13). Depressed
production of IL-2 (21, 45) and IFN-
(21, 40) in patients with chronic pulmonary tuberculosis has been reported. Similarly, IL-2 production was depressed in chronically
protein-deficient guinea pigs vaccinated with M. bovis BCG
(29), and TNF-
and IFN-
expression was reportedly
decreased in protein calorie-malnourished mice infected intravenously
with a lethal dose of the virulent M. tuberculosis Erdman
strain (6). In this study, the capacity of splenocytes and
macrophages to produce total bioactive IFN and TNF-
in vitro (Table
1) was found to be suppressed. The decreased production of these
cytokines at the site of immune inflammatory responses may be
responsible for the impairment of overall host antimycobacterial
resistance in protein-deficient guinea pigs. IFN-
is a key cytokine
in the development of Th1-type immune responses which are required for
the elimination of intracellular pathogens, including mycobacteria
(41, 48). IFN-
induces major histocompatibility class II
expression and thus increases antigen processing and presentation by
monocytes-macrophages to CD4 T lymphocytes (2). It also
induces differentiation and activation of monocyte-macrophages and
enhances their intracellular microbicidal activity (6).
Therefore, IFN-
is crucial in the immune response to mycobacterial
infection (9, 13). The present study revealed that total
bioactive IFN produced by immune splenocytes in protein-malnourished
guinea pigs was depressed. This decrease is most likely due to the
depressed production of IFN-
because protein malnutrition has been
observed to cause marked depletion of T lymphocytes (23, 28)
and profound depression of NK cell activity (20) in the
spleen, resulting in IFN-
-secreting cell loss and functional defect.
Moreover, IL-2 production, upon which IFN-
production is dependent,
was depressed in earlier studies with protein-malnourished guinea pigs
(29). All of these alterations induced by protein
malnutrition may contribute to the depressed production of IFN-
.
Dietary protein malnutrition impaired TNF-
production by peritoneal
macrophages. In contrast, intravenous injection of PPD triggered a
higher level of TNF-
in the serum of LP guinea pigs (Table 1). One
possible explanation is that vaccination with M. tuberculosis H37Ra may trigger inflammatory and immune responses in guinea pigs on the LP diet different from those in guinea pigs on
the HP diet. It is also possible that protein deficiency results in
leakage of lipopolysaccharide from the gut, which primes macrophages to
respond more dramatically to PPD in terms of TNF-
production.
TGF-
production could be influenced by many factors, including the
type and state of activation of cells, external stimuli, and the
presence of other cytokines. A study by Chandrasekar et al. revealed
that restricting dietary calories significantly increased mRNA and
protein expression of TGF-
and decreased those of proinflammatory cytokines, including IL-6 and TNF-
, in autoimmune prone mice (8). Bermudez reported that TGF-
production by human
monocyte-derived macrophages infected in vitro with M. avium
isolates was bacterial strain dependent and was more pronounced in
macrophages infected with a virulent strain (3). In this
study, we found that the serum of protein-deprived guinea pigs had
higher levels of bioactive TGF-
, while peritoneal macrophages from
protein-deficient guinea pigs had a depressed ability to produce
TGF-
in response to PPD (Fig. 4). Decreased TGF-
production in
response to PPD may reflect impairment of the ability of peritoneal
macrophages from protein-deficient guinea pigs to generate stimulating
components for TGF-
production. In fact, peritoneal macrophages from
protein-deficient guinea pigs produced significantly more
bioactive TGF-
when infected with virulent M. tuberculosis H37Rv in vitro for 4 days compared to
peritoneal macrophages from control diet guinea pigs (Fig. 5). There
was no difference between the diet treatment groups at 2 days (Fig. 5).
Given the slow replication rate of the bacilli, it is not surprising
that time in culture might be an important variable. One possible
explanation for the discrepancy between the effect of diet on the
production of TGF-
by macrophages exposed to PPD, in contrast to
viable M. tuberculosis, is that peritoneal macrophages from
protein-deficient guinea pigs cannot control the intracellular
multiplication of virulent mycobacteria as successfully as those from
control diet-fed animals, and thus mycobacteria grow faster and secrete
more proteins inside LP peritoneal macrophages. Some proteins
secreted by viable mycobacteria which are not necessarily found in
PPD could trigger TGF-
release from these infected macrophages.
Taken together, the results of this study demonstrate that protein
malnutrition has multiple detrimental effects on host antimycobacterial resistance. First, dietary protein malnutrition impairs macrophage functions, including TNF-
production and cooperation with T
lymphocytes to control mycobacterial growth in vitro; second, it causes
remarkable depression of T-lymphocyte functions, as indicated by
reductions in PPD-driven proliferation and Th1 cytokine (IFN-
)
production; third, protein malnutrition potentiates live M. tuberculosis H37Rv-infected monocytes-macrophages to produce
higher levels of TGF-
, which is a likely mediator of
immunosuppression and immunopathogenesis in tuberculosis (16, 17,
46).
 |
ACKNOWLEDGMENT |
This work was supported in part by PHS grant AI-15495 from NIH.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Immunology, College of Medicine, Texas A&M
University Health Science Center, College Station, TX 77843. Phone:
(409) 845-1367. Fax: (409) 845-3479. E-mail: dmcmurray{at}tamu.edu.
Editor: S. H. E. Kaufmann
 |
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