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*
Department of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106;
Department of Microbiology, National Institute of Respiratory Diseases, Mexico City, Mexico;
Bronchoscopy Service, National Institute of Respiratory Diseases, Mexico City, Mexico;
§
Department of Pathology, Case Western Reserve University, Cleveland, OH 44106; and
¶
Department of Tuberculosis Immunology, Statens Serum Institute, Copenhagen, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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activity. As the primary portal of entry
of M. tuberculosis is the lung, pulmonary immune
responses against multiple M. tuberculosis Ags were
compared between both M. tuberculosis-exposed tuberculin
skin test-positive healthy household contacts (HHC) of patients with
active sputum smear and culture-positive tuberculosis and tuberculin
skin test-positive healthy control individuals from the community (CC).
Frequencies of M. tuberculosis Ag-specific
IFN--producing cells, IFN- concentrations in culture
supernatants, and DNA synthesis in bronchoalveolar cells (BAC) and PBMC
were studied in HHC (n = 10) and CC
(n = 15). Using enzyme-linked immunospot assay we
found higher frequencies of IFN--producing cells with specificity to
M. tuberculosis-secreted Ag 85 (Ag 85) in BAC from HHC
than in BAC from CC (p < 0.022) and relative to
autologous PBMC, indicating compartmentalization of Ag 85-specific
cells to the lungs. Further, IFN--producing cells with specificity
to components A and B of Ag 85 were specifically compartmentalized to
the lungs in HHC (p < 0.05). IFN-
concentrations in culture supernatants of BAC and Ag-specific DNA
synthesis were low and comparable in the two subject groups. Increased
immune responses to Ag 85 at the site of repeated exposure to M.
tuberculosis (the lung) may represent an important component of
protective immunity against M. tuberculosis. Correlates
of protective immunity against M. tuberculosis are
required for assessment of the efficiency of anti-tuberculous
vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Active immune surveillance is required to maintain the latency of
quiescent M. tuberculosis foci, and
CD4+ T cells are critical to cell-mediated
anti-tuberculosis immunity (1, 2), presumably because
they are the primary source of the protective cytokine IFN-
(3, 4). The importance of IFN- responses in
mycobacterial infection is underscored by the observation that children
with hereditary IFN- receptor 1 deficiency are prone to infection
with ubiquitous mycobacteria or dissemination of Calmette-Guérin
bacillus after vaccination (5, 6, 7). Further, IFN- was
successfully used as adjunctive therapy in patients with refractory
nontuberculosis mycobacterial infection (8) and has shown
promising results in patients with multidrug-resistant pulmonary
tuberculosis (9).
In patients with pulmonary tuberculosis, however, M.
tuberculosis Ag-specific IFN--producing cells (10)
and spontaneous IFN- production (11) are
compartmentalized to the site of infection (the lung) despite ongoing
disease. Tuberculosis patients represent subjects who are at one end of
the spectrum of M. tuberculosis infection, i.e., individuals
who have lost immunological control, reactivated their infectious
focus, and developed disease. Tuberculosis patients, thus, may not
allow the study of protective immunity operative in the majority of
M. tuberculosis-infected individuals.
Healthy household contacts of patients with active tuberculosis
(HHC)5 are repeatedly
exposed to M. tuberculosis aerogenically. Under situations
of intense and prolonged exposure, the proportion of contacts found to
be infected with M. tuberculosis at the time of diagnosis of
sputum smear-positive tuberculosis index cases is as high as 30–40%
(12); however, infection rates can be even higher with
extended exposure to untreated patients with tuberculosis (up to 80%)
(13). The immunological study of M.
tuberculosis-reactive HHC therefore may allow elucidation of
protective immunity against M. tuberculosis. Because the
lung is the primary portal of entry for infection with M.
tuberculosis in humans, in the present study pulmonary immune
responses of tuberculin skin test-positive HHC to M.
tuberculosis Ags were compared with autologous systemic (blood)
immune responses and with pulmonary and systemic immune responses of
tuberculin skin test-positive healthy control individuals from the
community (CC). We found that IFN--producing cells with specificity
for Ag 85 complex (Ag 85co), which is considered to be a protective Ag
of M. tuberculosis, were compartmentalized to the lungs of
HHC, and that such cells were significantly more frequent in
bronchoalveolar cells (BAC) from HHC than in those from CC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subjects identified as HHC and CC who were willing to volunteer to undergo bronchoalveolar lavage, venipuncture, and HIV-1 serology were screened for participation in this study between 1996 and 1999 at the National Institute of Respiratory Diseases (Mexico City, Mexico). Approval to perform bronchoalveolar lavages, venipunctures, and HIV-1 serologies on consenting healthy individuals was given by the institutional review boards of National Institute of Respiratory Diseases and University Hospitals of Cleveland.
For recruitment of HHC, households of sputum smear-positive (grade 3)
patients with active tuberculosis who had been receiving
anti-mycobacterial chemotherapy no longer than 1 wk were chosen.
This was done under the assumption that shedding of M.
tuberculosis into the room air, and thus exposure of HHC to
M. tuberculosis, would be greater in these households than
in households of tuberculosis patients with lesser grades of smear
positivity (14). All HHC of the household who were willing
and met the following eligibility requirements were enrolled: 1) living
with the tuberculosis patient for a duration of 
3 mo, 2) apparent
clinical health, 3) normal chest radiograph, 4) tuberculin skin test
induration size >10 mm at 48–72 h upon injection of 5 tuberculin
units (TU) of purified protein derivative of M. tuberculosis
(PPD) by the Mantoux technique, 5) age 18–65 years, and 6) HIV-1
seronegativity.
CC were enrolled from staff at the National Institute of Respiratory Diseases and their relatives; none had any involvement in health care or known contact with patients with active tuberculosis. Eligibility requirements for the CC group were 1) apparent clinical health, 2) normal chest radiograph, 3) tuberculin skin test induration size >10 mm at 48–72 h upon injection of 5 TU PPD by the Mantoux technique, 4) age 18–65 years, and 5) HIV-1 seronegativity.
Exclusion criteria for HHC and CC were 1) blood hemoglobin concentration <10 g/dl, 2) any chronic systemic (diabetes, cancer) or pulmonary (asthma) disease, 3) upper respiratory tract infection within 1 mo before the study, and 4) immunosuppressive medications or immunosuppressive diseases.
After obtaining written informed consent from eligible HHC and CC, bronchoalveolar lavage (BAL) and venipuncture were performed in the morning on the day of each experiment.
Preparation of BAC
BAL was performed using an Olympus flexible fiberoptic bronchoscope as previously described (15). Briefly, after local anesthesia of the upper airways with 2–4% lidocaine, 150 ml of sterile isotonic saline was instilled into each of two segments of the right middle lobe. BAL fluid was centrifuged at 300 x g for 15 min at 4°C. BAC were counted and resuspended in complete medium (RPMI 1640 (BioWhittaker, Walkersville, MD) with 50 µg/ml gentamicin sulfate, 200 mM L-glutamine, and 10% heat-inactivated pooled human serum).
Preparation of PBMC
Between 40 and 60 ml of whole heparinized venous blood were obtained from each study subject. PBMC were prepared by centrifugation of the blood over Ficoll-Paque gradients (16) and were counted and resuspended in complete medium.
Antigens
PPD was purchased (Statens Seruminstitut, Copenhagen, Denmark).
Mannose-capped lipoarabinomannan (manLAM; purified from M.
tuberculosis strain H37Rv culture filtrate; endotoxin
contamination, 3.46 ng/mg manLAM, determined by Limulus
amebocyte assay) and Ag 85co purified from culture filtrate from H37Rv
and its three closely related components, Ag 85A, Ag 85B, and Ag 85C
(17), were provided by Dr. John T. Belisle (Department of
Microbiology, Colorado State University, Fort Collins, CO). Ag 85co is
a major secreted protein of metabolically active M.
tuberculosis, is fibronectin binding (18, 19), and
functions as a mycolyltransferase that is involved in the final stages
of mycobacterial cell wall assembly (20, 21). Secreted
protein from short term culture filtrate of M. tuberculosis,
ESAT-6 (endotoxin contamination, <0.05 ng/µg protein)
(22), and short term culture filtrate (ST-CF; endotoxin
contamination <0.05 ng/µg protein) (23) were additional
M. tuberculosis Ags used in this study. A nonmycobacterial
Ag prepared from Candida albicans (Candida; Greer
Laboratories, Lenoir, NC) was used to assess the specificity of
responses. All Ags were used in concentrations that resulted in peak
stimulation, as determined in previous studies (10). Dose
responses were determined for Ag 85A, Ag 85B, Ag 85C, ESAT-6, and ST-CF
for optimal induction of DNA synthesis, and performance of IFN- and
IL-10 enzyme-linked immunospot (ELISPOT) assays.
DNA synthesis
PBMC and BAC were added to round-bottom 96-well plates (Nunc, Copenhagen, Denmark) at 5 x 104 cells/well. Cells were stimulated with mycobacterial and nonmycobacterial Ags and the mitogen PHA (Sigma, St. Louis, MO). Complete medium was used as a negative control stimulus. Cultures were pulsed with 1 µCi/well [3H]thymidine (Amersham, Aylesbury, U.K.; sp. act., 5 Ci/mmol) on day 4, and cells were harvested 24 h later. Incorporation of [3H]thymidine was determined with a scintillation counter and expressed as counts per minute.
ELISAs
Twenty-four-hour supernatants from short term
cultures of PBMC and BAC that were stimulated with mycobacterial and
nonmycobacterial Ags were assessed by commercially available ELISA kits
for their content of IFN- (Endogen, Woburn, MA), and TNF-
(R&D
Systems, Minneapolis, MN).
ELISPOT assays
Because cytokine levels in cell culture supernatants reflect
production, degradation, and consumption by cells, ELISPOT analysis was
performed to determine the frequency of cytokine-producing cells and to
assess cytokine production on a single-cell basis. ELISPOT analysis is
10- to 200-fold more sensitive than ELISA (24).
Frequencies of cytokine-producing cells in PBMC and BAC were determined
by ELISPOT assay as previously described (25). Briefly, T
spot 96-well assay plates (Whatman, Clifton, NJ) were coated
overnight at 4°C with primary Abs against either IFN- (Endogen) or
IL-10 (PharMingen, San Diego, CA) at 2–10 µg/ml. Plates were washed
extensively and blocked with 1–2% BSA (Sigma). Then, PBMC and BAC
were added to the Ab-coated wells at a concentration of
105 cells/well and stimulated with mycobacterial
(PPD, manLAM, Ag 85co, Ag 85A, Ag 85B, Ag 85C, ESAT-6, and ST-CF) and
nonmycobacterial (Candida) Ags and PHA. Complete medium was
used as a negative control stimulus. Cultures were incubated at 37°C,
and cells were removed after 24 h (for IFN-) and 72 h (for
IL-10) by washing the wells three times with PBS. Biotinylated
secondary Abs to IFN- (Endogen, Woburn, MA) and IL-10 (PharMingen)
were then added to the wells, and plates were incubated at 4°C
overnight. Peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark;
1/2000) was added, and spots were visualized with 1%
3-amino-9-ethylcarbazole (Pierce, Rockford, IL).
The frequencies of IFN-- and IL-10-producing cells in each well were
determined using a computerized series 1 ImmunoSpot Image Analyzer
(Cellular Technology, Cleveland, OH). The average number of cytokine
spots in duplicate or triplicate wells was then calculated.
Statistical analysis
Comparisons between the HHC and CC groups were made using
nonparametric methods: Wilcoxon’s two-sample rank-sum test for
comparisons of distributions or Fisher’s exact test when
comparing proportions. In comparisons within groups examining
paired samples of lung vs blood, Wilcoxon’s signed-rank test (for
nonparametric data) was used. All statistical tests were conducted
using SigmaStat (version 2.0, Jandel, Chicago, IL) and SPSS for Windows
(version 8.0, SPSS, Chicago, IL) statistical software. Statistical
significance was set at p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten HIV-1-seronegative HHC (7 men and 3 women) and 15 HIV-1-seronegative CC (11 men and 4 women) underwent BAL and venipuncture to obtain BAC and PBMC. The median age of the HHC group (23.5 years) was not different from that of the CC group (24 years). An equal number (n = 4) of participants in each group were smokers. HHC were family members (spouse (n = 2), son (n = 2), daughter (n = 4), sister (n = 1), life partner (n = 1)) who lived in the same house (or the same room) with the index patient for a duration of at least 3 mo before the BAL.
The following median parameters were comparable between HHC and CC: body weight (62 and 67 kg), serum albumin (4.6 and 4.6 g/dl), peripheral white blood cell count (6.1 and 6.5 x 103/µl), and hemoglobin (16.3 and 15.9 g/dl), respectively. Median proportions of neutrophils were slightly higher in HHC than CC (63.8 and 56.1%, respectively; p = 0.045). In four of six HHC and seven of seven CC examined, scars indicating preceding vaccination with Calmette-Guérin bacillus were found. Tuberculin skin test indurations to 5 TU of PPD were comparable (20 and 18 mm in HHC and CC, respectively).
Retrieved proportions of BAL fluid, yield of BAC, and cellular profiles
of BAC, including proportions of peroxidase-positive cells (median, 2
and 1.5%) were comparable in HHC and CC, respectively. Proportions of
alveolar neutrophils were 1% in BAC of both HHC and CC. Median
proportions of alveolar lymphocytes were 14.1 and 14.0% and of
alveolar macrophages were 85.8 and 83.9%, in HHC and CC, respectively,
similar to those in CC in previous studies (10, 26).
DNA synthesis of BAC and PBMC in response to M. tuberculosis Ags
DNA synthesis (incorporation of
[3H]thymidine) was assessed in PBMC (Fig. 1
, A and C) and BAC
(Fig. 1, B and D) from HHC (A and
B) and CC (C and D) to optimal
concentrations of Candida (10 µg/ml), PPD (10 µg/ml), Ag
85co (5 µg/ml), ESAT-6 (2 µg/ml), ST-CF (1 µg/ml), manLAM (10
µg/ml), and PHA (1 µg/ml). As expected from the cell composition of
BAC in HHC and CC, DNA synthesis in response to all stimuli was low in
the two groups. DNA synthesis in response to PPD, Ag 85co, ST-CF,
Candida, and PHA was significantly lower
(p < 0.05) in BAC than in PBMC from both HHC
and CC. Interestingly, DNA synthesis in PBMC from HHC in response to
PPD, Ag 85co, and PHA was significantly lower than that in CC
(p < 0.05).
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The concentrations of Ags used for optimal induction of IFN-
spots were identical with those used for induction of DNA synthesis
(see above). PBMC and BAC from HHC (Fig. 2A) and CC (Fig. 2B) were stimulated with culture medium, PPD, and Ag 85co.
Fig. 2 depicts the frequencies of Ag-specific IFN--producing cells
(i.e., IFN- spots) in pairs of autologous PBMC and BAC of HHC and
CC. Due to the limited availability of lung cells, not all Ags could be
tested in all individuals.
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-producing cells was
significantly higher in BAC from HHC (n = 10) than in
BAC from CC (n = 13; p = 0.022). The
frequencies of PPD-specific IFN--producing cells were not
statistically different in BAC of HHC compared with those of CC.
Further, the frequencies of PPD- and Ag 85co-specific IFN--producing
cells were higher in BAC than in autologous PBMC from six and seven
HHC, respectively. In contrast, in CC, the frequencies of PPD- and Ag
85co-specific IFN--producing cells were lower in BAC than in
autologous PBMC in 7 of 15 CC tested for PPD and in 7 of 13 CC tested
for Ag 85co. When a cutoff of 3 was chosen for the fold increase in
frequencies of IFN--producing cells in BAC compared with that in
autologous PBMC, the frequencies of IFN--producing cells in BAC of
HHC (n = 10) were 3-fold higher than those in
autologous PBMC in 40% to PPD and in 50% to Ag 85co. Using the same
criteria, the frequencies of IFN--producing cells in BAC of CC were
higher than those in autologous PBMC in 7% to PPD (1 of 15) and in
12% to Ag 85co (1 of 13; p = 0.05 for Ag 85co, HHC vs
CC). Thus, M. tuberculosis-specific IFN--producing cells
appeared to be enriched in the lungs (compared with the blood) of HHC
and were present at higher frequencies in BAC from HHC than in those
from CC. In only a limited number of CC (n = 2) were
the frequencies of PPD- and Ag 85co-specific IFN--producing BAC
higher than those in PBMC, indicating possible exposure to M.
tuberculosis within the community.
To assess whether increased frequencies of IFN--producing
Ag-specific cells were due to differences in absolute numbers of
lymphocytes within BAC or PBMC of HHC and CC, frequencies of
IFN--producing cells were normalized to 10,000 lymphocytes in BAC
and PBMC, respectively. Median numbers of Ag 85-specific
IFN--producing cells per 10,000 lymphocytes in BAC and PBMC were 29
(25th percentile, 17; 75th percentile, 90) and 7 (25th percentile, 2;
75th percentile, 11), and 13 (25th percentile, 5; 75th percentile, 34)
and 6 (25th percentile, 2; 75th percentile, 14) in HHC and CC,
respectively. Median numbers of PPD-specific IFN--producing cells
per 10,000 lymphocytes in BAC and PBMC were 56 (25th percentile, 39;
75th percentile, 148) and 17 (25th percentile, 9; 75th percentile, 23)
and 34 (25th percentile, 23; 75th percentile, 92) and 8 (25th
percentile, 7; 75th percentile, 27) in HHC and CC, respectively.
Differences in frequencies of Ag 85-specific IFN--producing cells
per 10,000 lymphocytes in BAC and PBMC were significant in HHC only
(p = 0.022).
To estimate the output of IFN- from IFN--producing cells in PBMC
and BAC from HHC and CC, sizes of spots were measured with the image
analyzer. Proportions of IFN- spots from Ag 85co-stimulated PBMC and
BAC of HHC (n = 8) and CC (n = 8)
within each size category
(10-1–10-3
mm2) were comparable between BAC and PBMC from
HHC and CC, indicating comparable outputs of IFN- per cell (Fig. 3).
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-producing PBMC and BAC were studied in subgroups
of HHC (n = 6) and CC (n = 6). Median
frequencies of ST-CF-specific IFN--producing cells per
105 BAC and PBMC in HHC and CC paralleled those
of PPD responses and were 2-fold higher in BAC than in PBMC in four of
six HHC and in zero of six CC (HHC BAC, 147 (25th percentile, 16; 75th
percentile, 206); HHC PBMC, 51 (25th percentile, 34; 75th percentile,
101); CC BAC, 38 (25th percentile, 24; 75th percentile, 68); CC PBMC,
85 (25th percentile, 43; 75th percentile, 139); not significant).
Median frequencies of ESAT-6-, manLAM-, and Candida-specific
IFN--producing cells were low (<30/105 PBMC
or BAC) in both HHC and CC. On the other hand, median frequencies of
PHA-induced IFN- spots were high (580–810/105
PBMC or BAC) and comparable in both HHC and CC (data not shown).
In a subset of study subjects (HHC, n = 6; CC,
n = 6) frequencies of IFN--producing cells were also
assessed in response to the three components of Ag 85co, namely, Ag 85A
(5 µg/ml), Ag 85B (5 µg/ml), and Ag 85C (5 µg/ml; Table I). The ratios of the frequencies of Ag
85A- and Ag 85B-specific, but not of Ag 85C-specific, IFN--producing
BAC and PBMC (BAC/PBMC) were significantly higher in HHC than in CC
(p = 0.01).
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IFN-- and TNF- concentrations by ELISA
In a subgroup of study subjects (HHC, n = 6; CC,
n = 8), 24-h culture supernatants from in vitro Ag
85co- and PPD-stimulated PBMC and BAC were available for analysis of
IFN- and TNF- concentrations by ELISA. IFN- production in
response to Ag 85co was significantly lower in BAC from HHC than in BAC
from CC (p = 0.032). Thus, high frequencies of
Ag 85co-specific IFN--producing cells in BAC from HHC were not
accompanied by a corresponding higher concentration of IFN- in cell
culture supernatants.
Ag 85co-induced TNF- production was significantly higher in BAC
compared with PBMC in both HHC and CC. In HHC, the median TNF- level
in BAC was 6871 pg/ml (25th percentile, 4669; 75th percentile, 7513),
and in PBMC it was 2457 pg/ml (25th percentile, 1537; 75th percentile,
3282; p = 0.043). In CC the TNF- level in BAC was
3187 pg/ml (25th percentile, 2906; 75th percentile, 4113), and in PBMC
it was 1688 pg/ml (25th percentile, 1377; 75th percentile, 1853;
p < 0.043). TNF- concentrations were higher
(1.8-fold) in Ag 85co-stimulated BAC from HHC than in those from CC
(not significant).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing BAC were compartmentalized to the alveolar spaces.
Further, frequencies of Ag 85co-specific, IFN--producing cells in
BAC from HHC were higher than those in BAC from CC. Also, frequencies
of Ag 85A- and Ag 85B-specific, but not of Ag 85C-specific,
IFN--producing cells were significantly higher in BAC from HHC than
in those from CC. Considering that the proportions of lymphocytes in
alveolar spaces of healthy subjects (i.e., both HHC and CC) are 4- to
5-fold lower than that in peripheral blood, the expansion of Ag
85co-specific IFN--producing cells in BAC from HHC to proportions
higher than those in PBMC is notable. These findings are consistent
with aerogenic M. tuberculosis infection/exposure in HHC of
patients with tuberculosis. Thus, it appears that during the early
phases of M. tuberculosis infection, Ag 85co-specific
pulmonary mononuclear cells are expanded.
Ag 85co is a predominant product of live M. tuberculosis
(19, 27) and may be critical in the development of
protective immunity in M. tuberculosis infection
(28). Recently, it has been shown that vaccination with Ag
85co conferred protection against M. tuberculosis challenge
in mice (29, 30, 31) and guinea pigs (32, 33). In
studies of human immune responses to Ag 85, PBMC from healthy
tuberculin skin test-positive subjects demonstrated strong DNA
synthesis and IFN- secretion in response to both Ag 85co and Ag 85A
(34). Further, IFN- production of Ag 85-stimulated PBMC
from HHC was significantly higher than that of PBMC from patients with
tuberculosis (35). In fact, DNA synthesis of PBMC in
response to Ag 85co was low in 52% (33, 35) to 100% of
patients with active tuberculosis (36) relative to that in
healthy control individuals. Further, IFN- production in response to
Ag 85A was low in patients with pulmonary tuberculosis (34, 37). Overall, it appears that T cell responses to Ag 85co may be
indicative of host protective immunity against M.
tuberculosis in humans. Our finding of increased frequencies of Ag
85co-specific IFN--producing BAC in tuberculin skin test-positive
HHC may indicate the expansion of local protective immunity in these
subjects who have a high probability of having been infected with
M. tuberculosis.
The observed differences between frequencies of Ag 85-specific
IFN--producing BAC in HHC and CC are unlikely to be due to
differences in the composition of blood and lung cells, as no
differences in number or percentage of lymphocytes and mononuclear
phagocytes were found by histochemical analysis of BAC and PBMC from
HHC and CC. In particular, proportions of lymphocytes were comparable
between the study groups. Additionally, frequencies of
IFN--producing cells that were specific for M.
tuberculosis Ags other than Ag 85co (PPD, ST-CF, manLAM, ESAT-6),
were comparable in BAC from both study groups. Thus, the increased
proportions of Ag 85co-specific IFN--producing BAC in HHC probably
resulted from expansion of Ag 85co-specific BAC that occurred in
response to recent aerogenic M. tuberculosis
infection/exposure in the households of tuberculosis patients. This is
consistent with the finding in HHC that when frequencies of Ag-specific
IFN--producing cells were normalized to 10,000 lymphocytes in both
PBMC and BAC, numbers of PPD- and Ag 85-specific IFN--producing
cells were significantly higher in lymphocytes in BAC than in
lymphocytes in PBMC. However, because recent data indicate that
M. tuberculosis-infected AM might be sources of IFN-
(38), normalization of IFN--producing cells to numbers
of lymphocytes in BAC may not reflect the true range of
IFN--producing cell populations in BAC. It was beyond the scope of
this study to unravel the cellular source of IFN- and to assess the
influence of exposure to M. tuberculosis on the cell
populations involved in production of IFN-. Elucidation of the
cellular source of IFN- in HHC and CC is the subject of ongoing
research.
Compartmentalization of Ag 85co-specific IFN--producing BAC in lungs
from HHC was detectable only by a highly sensitive assay (ELISPOT) and
not by DNA synthesis or IFN- immunoreactivity in supernatants from
cell cultures.
The implications of increased frequencies of M.
tuberculosis-specific IFN--producing cells in the lungs of HHC
are not presently clear, as IFN--producing M.
tuberculosis Ag-specific BAC are also detectable in high
frequencies in BAC from radiographically affected areas of lungs from
tuberculosis patients (10).
Time points of conversion of tuberculin skin tests from negative to
positive could not be assessed in HHC due to the cross-sectional nature
of this study. Definitive diagnosis of new infection or reinfection
with M. tuberculosis in HHC as a result of contact with the
tuberculosis patients, therefore, was not possible. Several
observations, however, might be indicative of recent exposure to or
infection with M. tuberculosis in HHC in this study. First,
slightly increased proportions of circulating neutrophils in the
peripheral blood might indicate a systemic and recent inflammatory
response. Further, TNF- production by BAC from HHC in response to Ag
85co was increased (although the difference from CC did not reach
statistical significance) and might indicate local inflammatory immune
responses. Lastly, lower levels of DNA synthesis in PBMC of HHC
compared with CC in response to PPD, Ag 85co, and PHA might suggest the
activation of immunosuppressive circuits due to recent active M.
tuberculosis infection, as also seen with other infections
(39).
The frequencies of IFN--producing ST-CF-specific cells were higher
in BAC than in PBMC in the majority of HHC, but were not significantly
different from those in BAC of CC. These differences may, however,
reach significance if larger populations are studied. ST-CF is a crude
mixture of secreted Ags and contains Ag 85co and a multitude of other
Ags. Also, since the concentration of purified Ag 85co used in the
current study (5 µg/ml) exceeded that in crude ST-CF (at most 1
µg/ml), differences in the detectable frequencies of
IFN--producing Ag 85co- and ST-CF-specific cells may have resulted
from suboptimal stimulation of cells by the latter Ag mixture.
Alternatively, ST-CF may contain components with suppressive activities
for expression of IFN-. Demissie et al. have recently shown that
ST-CF induces greater DNA synthesis and higher IFN- production in
PBMC from HHC compared with those from patients with tuberculosis
(40). Interestingly, levels of IFN- production were
significantly higher in ST-CF-stimulated PBMC from HHC of patients with
advanced tuberculosis compared with those from HHC of patients with
minimal disease, suggesting an association with more intense exposure
to M. tuberculosis.
We found that the frequencies of IFN--producing BAC with specificity
for the purified components of Ag 85co, namely, Ag 85A and Ag 85B, but
not Ag 85C, were significantly higher in BAC of HHC than in BAC of CC,
resembling the findings with the purified Ag 85co. The relative
immunogenicity of the components of Ag 85co (A, B, and C) are not known
to differ despite the predominance of Ag 85B in purified Ag 85co. Also,
whether there are differences among Ag 85A, -B, and -C with regard to
biological activity and/or induction of protective immunity is not
known.
In conclusion, recent aerogenic exposure/infection with M.
tuberculosis is associated with the accumulation of Ag
85co-specific IFN--producing cells in the alveolar spaces of HHC.
The finding of compartmentalization of Ag 85co-specific
IFN--producing cells to the alveolar spaces of HHC may reflect the
activation of early protective immune responses in situ. The
understanding of human protective immune responses to M.
tuberculosis at sites of infection may facilitate the
identification of correlates of protective immunity, which are urgently
needed in the evaluation of the efficacy of new anti-tuberculosis
vaccines.
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Acknowledgments |
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Footnotes |
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2 Excerpts of the data were presented at the 34th U.S.-Japan Conference on Tuberculosis/Leprosy, San Francisco, CA, June 27–30, 1999.
3 Address correspondence and reprint requests to Dr. Stephan Schwander, Department of Medicine, Division of Infectious Diseases, Case Western Reserve University, Biomedical Research Building, Room 1001, 10900 Euclid Avenue, Cleveland, OH 44106-4984.
5 Abbreviations used in this paper: HHC, healthy household contacts of patients with active tuberculosis; CC, tuberculin skin test-positive healthy control individuals from the community; Ag 85co, Ag 85 complex; BAC, bronchoalveolar cells; BAL, bronchoalveolar lavage; PPD, purified protein derivative of M. tuberculosis; TU, tuberculin units; manLAM, mannose-capped lipoarabinomannan; ST-CF, short term culture filtrate; ELISPOT, enzyme-linked immunospot.
Received for publication December 9, 1999. Accepted for publication May 16, 2000.
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References |
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in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249. gene-disrupted mice. J. Exp. Med. 178:2243.-receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335:1941.-receptor deficiency in an infant with fatal bacille Calmette-Guerin infection. N. Engl. J. Med. 335:1956. receptor 1 deficiency in a child with tuberculoid bacillus Calmette-Guerin infection and a sibling with clinical tuberculosis. J. Clin. Invest. 100:2658.[Medline]
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-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes. J. Immunol. 163:2267. interferon production, and serum immunoglobulin G directed against a purified 32-kDa mycobacterial protein antigen (P32) in patients with active tuberculosis. Scand. J. Immunol. 27:187.[Medline]
interferon production in human alveolar macrophages by Mycobacterium tuberculosis. Infect. Immun. 65:5149.[Abstract]
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