Divisions of Pulmonary/Critical Care Medicine and Infectious Diseases, Department of Medicine, and Department of Surgery, Indiana University Medical Center, Indianapolis, Indiana 46202
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ABSTRACT |
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A
CD8+ lymphocytic alveolitis occurs
in up to 60% of asymptomatic human immunodeficiency virus
(HIV)-infected individuals. Early in HIV infection, lymphocytes consist
predominantly of cytotoxic T lymphocytes directed against HIV-infected
targets. As HIV disease progresses, they are replaced by
CD8+CD57+
suppressor cells. Virus-specific cytotoxic T
lymphocytes secrete interferon- (IFN-
), an important cytokine in
upregulating immune responses, primarily through macrophage activation.
We examined the ability of lung and blood lymphocytes from HIV-positive
patients at various stages of HIV infection to secrete IFN-
spontaneously and in response to phytohemagglutinin A. IFN-
production and secretion were determined with ELISA, Western
blot, immunoprecipitation, and Northern blot techniques. Lung
lymphocytes from HIV-infected individuals secreted large amounts of
IFN-
. However, this ability was lost in patients with late-stage
disease. Correlation between blood and lung lymphocyte IFN-
secretion was poor, suggesting regional differences in lymphocyte
function. These data suggest that lung levels of IFN-
are high until
late in HIV disease. These findings support the concept of
administering exogenous IFN-
to patients with late-stage HIV disease
and opportunistic infections.
lymphocytic alveolitis; cytotoxic T lymphocytes; suppressor cells; macrophage activation; human immunodeficiency virus
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INTRODUCTION |
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STUDIES INDICATE THAT alveolar macrophages (AMs) and lymphocytes in the lower respiratory tract of human immunodeficiency virus (HIV)-infected individuals are activated (4, 40). The latter cells have been shown to accumulate in the alveolar space, resulting in lymphocytic alveolitis. Throughout most of the course of HIV infection, alveolar lymphocytes consist predominantly of CD8+ cytotoxic T lymphocytes (CTLs) (1, 12). Because these CTLs are directed against HIV antigens (25), it is reasonable to hypothesize that lymphocytic alveolitis represents an appropriate local immune response against HIV-infected lung cells.
The initiation of a CTL response requires interaction between T cells
and accessory cells (in the alveolar space represented by AMs) that
have been exposed to antigen. Prior investigations (34-36) in our
laboratory have shown that AM accessory cell function is enhanced in
HIV-infected patients, likely due to augmented secretion of AM
cytokines important in T-cell proliferation. As a result, T cells are
induced to express high-affinity interleukin (IL)-2 receptors and
secrete IL-2 (13, 14), a factor necessary for lymphocyte proliferation
and differentiation of precytotoxic T lymphocytes into CTLs (38). Spain
et al. (32) previously showed that lung lymphocytes from asymptomatic
HIV-infected patients with lymphocytic alveolitis proliferate
spontaneously and secrete IL-2, thereby demonstrating the potential for
autonomous in situ lymphoproliferation within the lungs of these
individuals. Interestingly, simultaneous experiments demonstrated
almost no IL-4 secretion, suggesting that these cells secreted a
Th1-like cytokine profile (33). Other investigators (10) have shown
that murine CD8+ CTLs secrete
cytokines consistent with a Th1 pattern. Interferon- (IFN-
) is
another Th1 cytokine and, in fact, has been shown to be
released by peripheral blood CTLs from HIV-infected individuals on
encountering target antigens (15). However, studies (20, 22, 23, 27)
examining lymphocyte IFN-
production in HIV infection have yielded
conflicting results, likely due to the inclusion of patients at
different stages of disease progression.
The presence of activated AMs and CTLs in the alveolar space of
HIV-infected individuals suggests that IFN- may be actively secreted
by alveolar T cells. In this study, we analyzed IFN-
production and
secretion by lung and peripheral blood lymphocytes in patients at
various stages of HIV infection.
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MATERIALS AND METHODS |
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Subjects. Seventeen HIV-positive patients (mean age 36.4 ± 7.5 yr; 16 men, 1 woman) served as the study population. Nine were nonsmokers and eight were current or ex-smokers. All HIV-positive individuals had no current pulmonary symptoms and underwent bronchoscopy only for the purposes of this study. Chest radiographs were obtained on the day of bronchoscopy if one had not been performed in the previous 3 mo and were normal in all cases. According to the 1993 revised Centers for Disease Control and Prevention classification system for HIV infection (5), six were in class A1, five in A2, one in B2, two in B3, and three in C3. The mean CD4+ T-cell count of the population was 396 ± 67 cells/µl (range 3-870 cells/µl). HIV-positive subjects were further divided into three groups based on peripheral blood CD4 counts: >500/µl (n = 6), 200-500/µl (n = 6), and <200/µl (n = 5). Four nonsmoking normal adults (mean age 26 ± 2 yr; 1 man, 3 women) were also studied.
Bronchoalveolar lavage. After the upper airways were anesthetized with 2% topical lidocaine, bronchoalveolar lavage (BAL) was performed through a fiber-optic bronchoscope wedged in subsegmental bronchi in the right middle and right lower lobes. Room temperature normal saline (100 ml) was instilled in 20-ml aliquots into three separate bronchi. Typically, 250-300 ml were instilled to obtain a return of 125-200 ml. Recovered lavage fluid was kept on ice until processed. HIV-positive patients yielded an average of 15.1 ± 2.4 × 106 bronchoalveolar cells, of which 33.8 ± 5.8% were lymphocytes by morphological criteria. Normal volunteers yielded a mean of 10.3 ± 4 × 106 cells, of which 30 ± 1% were lymphocytes. Note that the normal subjects represent a selected group of individuals with an unusually high percentage of lymphocytes within the alveolar space to ensure that enough lymphocytes could be isolated for study.
Preparation of lung and peripheral blood T
cells. Lavage fluid was filtered through 100-µm nylon
mesh (Tetko, Elmsford, NY) to remove debris and centrifuged at 400 g for 10 min. The cell pellet was
washed twice in Hanks' balanced salt solution and
resuspended in RPMI 1640 medium with 25 mM HEPES
(GIBCO, Grand Island, NY) supplemented with 5% heat-inactivated FCS
(GIBCO), 24 µg/ml of gentamicin, 100 U/ml of
penicillin G, 100 µg/ml of streptomycin, 250 ng/ml of amphotericin
B, and 2 mM
L-glutamine. Cell viability was
determined by 1% trypan blue exclusion and was routinely >95%. Immediately after bronchoscopy, peripheral blood was collected in a
heparinized syringe for isolation of peripheral blood T
cells. Peripheral blood mononuclear cells (PBMCs) were
separated by centrifugation through a Ficoll-Hypaque gradient. PBMCs
and bronchoalveolar cells were cultured on plastic for 1 h at 37°C
to remove adherent monocytes. Nonadherent cells were subsequently
passed over a nylon wool column to remove residual monocytes
and/or macrophages and B cells (17). The resultant population
consisted of 97% lymphocytes as determined by morphological
criteria. More than 70% of these cells were
CD3+. In preliminary studies,
immunofluorescent staining demonstrated that HIV lung lymphocytes had a
CD4-to-CD8 cell ratio of 0.57, normal lung lymphocytes had a CD4-to-CD8
cell ratio of 1.16, and HIV blood lymphocytes had a CD4-to-CD8 cell
ratio of 0.40. These percentages are similar to those reported by other
investigators (40). In some experiments, nonadherent BAL cells and
PBMCs were further purified into
CD8+ and
CD4+ subsets with the use of
immunomagnetic beads (Dynal, Lake Success, NY). Briefly, lymphocytes
were incubated with magnetic beads coated with anti-CD8 at a 4:1
bead-to-cell ratio. Cells were positively selected for with a magnet.
Nonadherent cells were incubated with beads coated with anti-CD4 to
obtain a purified CD4+ subset.
CD4+ cells isolated in this manner
were >96% CD3+, >95%
CD4+, and <1%
CD8+. Similarly, the
CD8+ population was >93%
CD3+, >96%
CD8+, and <1%
CD4+. Because of the limited
number of lung lymphocytes that could be isolated from a single
volunteer, not all experiments were performed in every subject. More
than 95% of the isolated cell populations were viable by the trypan
blue exclusion test.
Measurement of secreted IFN-, IL-4,
and IL-10 by ELISA. Lung and blood lymphocytes were
readjusted to a concentration of 1 × 106 viable cells/ml in complete
medium supplemented with 10% human serum. Cells were cultured in
96-well flat-bottomed tissue culture plates (Costar, Cambridge, MA) at
37°C in the presence and absence of 1 µg/ml of phytohemagglutinin
A (PHA; Wellcome Diagnostics, Research Triangle Park, NC). After 48 h,
supernatants were harvested, centrifuged to remove cells and debris,
and stored at
70°C until further use. IFN-
secretion was
measured with a commercially available ELISA (Endogen, Boston, MA) with
a sensitivity of 5 pg/ml. IL-4 and IL-10 were measured with a sandwich
protocol as previously described (39). The sensitivities of these
assays are 78 pg/ml for IL-4 and 3.9 ng/ml for IL-10.
Measurement of IFN- production by
Western blot and immunoprecipitation. For Western
blotting, supernatants were mixed with Laemmli buffer [0.08%
SDS, 0.7 M 2-mercaptoethanol, 1 M glycerol, 0.06 M Tris
(pH 6.8), and 0.05% bromphenol blue], and proteins were resolved
by SDS-PAGE on a 12% polyacrylamide gel. Proteins were electroblotted
to nitrocellulose, nonspecific binding sites were blocked with blocking
buffer (GIBCO) for 4 h, and the membrane was incubated overnight with
polyclonal rabbit anti-human IFN-
antibody at a 1:100 dilution at
4°C. After the membrane was washed, it was then incubated with a
1:3,000 dilution of goat anti-rabbit IgG-peroxidase for 1 h at room
temperature. Signal was then detected by enhanced chemiluminescence
techniques with a commercially available kit (Amersham Life Sciences,
Arlington Heights, IL).
For immunoprecipation experiments, lung and blood lymphocytes were
resuspended at 2 × 106
cells/ml and cultured for 48 h in methionine-free complete medium supplemented with 10% human serum and 100 µCi/ml of
[35S]methionine
(specific activity 800 Ci/mmol; NEN, Boston, MA) in the presence and
absence of 1 µg/ml of PHA. Supernatants were harvested, and protease
inhibitors were added to the supernatants for a final concentration of
(in mM) 3 phenylmethylsulfonyl fluoride, 5 N-ethylmaleimide, 5 EDTA, and 2 p-amino- benzamidine
hydrochloride. After samples were precleared by
incubation with normal rabbit serum (Sigma) and a 10% protein A cell
suspension (Sigma), polyclonal rabbit anti-human IFN- (Genzyme,
Cambridge, MA) was added to each sample and incubated overnight at
4°C. Nonimmune rabbit serum was added to some samples as a negative
control. Antigen-antibody complexes were precipitated for 2 h at
37°C with protein A-Sepharose (Sigma) and then isolated by
centrifugation. The pellet was washed five times, resuspended in
Laemmli buffer, heated at 100°C for 5 min, and centrifuged at
10,000 g for 5 min, and supernatants were analyzed by SDS-PAGE on a 15% polyacrylamide-1.7% bisacrylamide gel with a 3.3% polyacrylamide-1.7% bisacrylamide
stacking gel. Autoradiography was then performed as previously
described (34).
Measurement of IFN- gene
transcription. Whole cell RNA was isolated by the
guanidine isothiocyanate method of Chomczynski and Sacchi (6) from
freshly isolated lung and blood lymphocytes and from cells that had
been cultured at 106 cells/ml for
16 h in the presence of 1 µg/ml of PHA and 2 ng/ml of phorbol
12-myristate 13-acetate. Equal amounts of RNA (2 µg) were fractionated on a 1.5% denaturing agarose gel containing 2.2 M
formaldehyde by the method of Lehrach et al. (18).
Escherichia coli 23S and 16S mRNAs
(Pharmacia Fine Chemicals, Piscataway, NJ) served as standards. RNA was
transferred to Gene Screen Plus (NEN) and baked at 80°C for 2 h.
The IFN-
probe was prepared by isolating a 1-kb
Pst I fragment of the p52 plasmid
obtained from the American Type Culture Collection (Manassas, VA) (7). The probe was 32P labeled with an
oligo-labeling kit (Pharmacia). Membranes were prehybridized in 50%
formamide, 1 M NaCl, 10% dextran, 0.05 M Tris, 1% SDS, 1×
Denhardt's solution, and 100 µg/ml of denatured salmon testes DNA
for 5 h. Hybridization was performed in fresh prehybridization fluid
containing 106
counts · min
1 · ml
1
labeled probe. After hybridization, blots were washed and exposed to
XAR-2 film.
Statistics. Comparisons between HIV
subgroups and between HIV-infected and normal populations were made
with the Mann-Whitney rank sum test for nonparametric data. Comparisons
between blood and lung T cells were done with a paired
t-test.
P 0.05 was considered significant.
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RESULTS |
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Secretion of IFN- by lung and blood
lymphocytes. Prior experiments in our laboratory
demonstrated that lung lymphocytes from asymptomatic HIV-infected
individuals with lymphocytic alveolitis secreted IL-2 but minimal IL-4,
indicating that these cells secreted a Th1-like cytokine profile (32).
Because IFN-
is another Th1 cytokine, we hypothesized that its
secretion would also be upregulated. Lung lymphocytes were isolated
from normal volunteers and HIV-infected patients, and their ability to
secrete IFN-
spontaneously and in response to PHA was measured with
an ELISA. Figure 1 demonstrates that lung
lymphocytes from HIV-infected patients and normal volunteers secreted
small amounts of IFN-
spontaneously. After stimulation with PHA,
lung lymphocytes from both groups secreted significantly more IFN-
than unstimulated cells. However, there was no difference between the
HIV-infected and normal populations. We found no relationship between
CD4-to-CD8 cell ratios or the absolute percentage of
CD4+ or
CD8+ cells in blood and lung
lymphocyte preparations and IFN-
secretion, suggesting that both
T-cell populations were producing IFN-
.
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In two subjects, IFN- was not secreted by lung or blood lymphocytes.
In the remaining individuals, correlation between blood and lung
lymphocyte IFN-
secretion was poor, suggesting regional differences
in lymphocyte function (Fig. 2). In some
subjects, PHA-stimulated lung lymphocytes secreted more IFN-
than
autologous blood lymphocytes, suggesting a compartmentalized pulmonary
response. However, this was not a universal finding
because in other subjects, blood lymphocytes secreted
more IFN-
than autologous lung lymphocytes.
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CD4+ lymphocytes have
traditionally been thought of as the major producers of IFN- (29),
and, indeed, some investigators have found increased IFN-
production
early in HIV infection before depletion of
CD4+ cells (27). Recent studies
(10, 15) have shown that CD8+ CTLs
also produce IFN-
when they encounter target antigens. Thus
experiments were performed to determine which lymphocyte subsets were
actively secreting IFN-
in the lungs and blood of HIV-infected
subjects. Purified lymphocyte subsets were isolated by immunomagnetic
separation techniques and analyzed for spontaneous IFN-
secretion.
The results from two experiments shown in Table 1 demonstrate that
CD8+ lymphocytes are major
producers of IFN-
in the lungs and blood of HIV-infected
individuals. These experiments demonstrate that lung
CD8+ cells secrete as much
IFN-
, if not more, than CD4+
cells. Furthermore, in these two experiments, lung
CD8+ cells tended to secrete more
IFN-
than blood CD8+ cells,
again suggesting a compartmentalized pulmonary response.
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Western blot and immunoprecipitation
experiments. To confirm the ELISA results, Western
blotting was performed. A representative of three blots is shown in
Fig. 3. In this experiment, both
CD4+ and
CD8+ lung lymphocytes
spontaneously secreted IFN- as demonstrated by the 17-kDa band
corresponding to the molecular mass of mature IFN-
,
which may reflect spontaneous cell activation through excessive manipulation or magnetic beads. IFN-
secretion was slightly greater when lymphocytes were stimulated with PHA. Secretion of IFN-
by
CD8+ lung lymphocytes was greater
than the corresponding peripheral blood population. Immunoprecipitation
studies demonstrated that PHA-stimulated lung lymphocytes actively
synthesized IFN-
. Furthermore, immunoprecipitation studies on T-cell
subsets again demonstrated that IFN-
was actively produced by both
CD4+ and
CD8+ blood and lung lymphocytes
(data not shown).
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Northern blots. Steady-state IFN-
mRNA levels were determined through Northern blot analysis. Figure
4 depicts IFN-
mRNA concentrations in
freshly isolated PHA-phorbol 12-myristate
13-acetate-stimulated lung and blood lymphocytes from
an HIV-infected individual. Stimulated lung lymphocytes
contained more message for IFN-
than similarly stimulated blood
lymphocytes. Interestingly, a faint signal for IFN-
message was seen
in unstimulated freshly isolated lung lymphocytes but not in blood
lymphocytes. The presence of IFN-
mRNA in freshly isolated lung
lymphocytes suggests there is ongoing production of IFN-
in the
lungs of HIV-infected individuals.
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Changes in IFN- secretion with disease
progression. The
CD8+ lymphocyte subset contains
two major populations of cells: CTLs and suppressor cells. Other
investigators (16, 28) have demonstrated that with HIV disease
progression, CTLs in the alveolar space are replaced by
CD57+-expressing suppressor cells.
These latter cells may have different abilities to produce and secrete
IFN-
. Figure
5A shows
lung lymphocyte IFN-
secretion by three different subsets of
HIV-infected subjects as defined by the peripheral blood CD4 count.
Lung lymphocytes from patients with early- and late-stage HIV infection
produced significantly less IFN-
than lymphocytes from subjects with
CD4 counts between 200 and 500 cells/µl. Similar trends were seen in
the vascular compartment, although the ability of blood lymphocytes from patients at differential stages of HIV infection to secrete IFN-
did not reach significance (Fig.
5B). These data suggest that in patients with late-stage disease, the ability of lung lymphocytes to produce and secrete IFN-
is lost. This does not necessarily indicate a shift from Th1-like to Th2-like cells. In no
instance did lung lymphocytes from patients with CD4 counts <200
cells/µl secrete IL-4 or IL-10 (n = 4 patients). In fact, IL-4 secretion was detectable in low amounts only
in PHA-stimulated lung lymphocytes from patients with CD4 counts
between 200 and 500 cells/µl (mean 340 pg/ml;
n = 5 patients).
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DISCUSSION |
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In this study, we investigated secretion of IFN- by lung and blood
lymphocytes in HIV-positive individuals at various stages of disease
progression who have lymphocytic alveolitis. All subjects were without
pulmonary symptoms and had no active disease on chest radiographs.
Previous studies (1, 12, 25) have demonstrated that in early HIV
infection, alveolar lymphocytes consist predominantly of
CD3+/CD8+
CTLs directed against HIV-infected cells. Peripheral blood
CTLs, including those directed against HIV-infected targets, have been shown to secrete IFN-
(10, 15). Our study extends these findings to
the tissue level by demonstrating that lung lymphocytes from HIV-infected individuals are fully capable of secreting large amounts
of IFN-
. The ability to secrete IFN-
is associated with increased
IFN-
mRNA levels and subsequent active protein synthesis. Correlation between blood and lung lymphocyte IFN-
is poor,
suggesting that regulation of IFN-
production differs in the
pulmonary and vascular compartments. However, in some individuals, lung
lymphocytes secrete more IFN-
than autologous blood lymphocytes,
suggesting an augmented ability to produce and secrete IFN-
at the
tissue level. Finally, we demonstrated that the ability of alveolar
lymphocytes to secrete IFN-
is most pronounced in HIV-infected
subjects in the middle stages of their disease. Early in HIV infection,
when pulmonary complications are uncommon, IFN-
secretion by lung lymphocytes is similar to that in HIV-negative subjects, with elevated
lymphocytes in BAL. More importantly, late in HIV infection, IFN-
levels decline, providing a theoretical explanation for the increased prevalence of opportunistic infections in late-stage HIV
disease. The difference in IFN-
secretion among the three subgroups
is much less pronounced in blood lymphocytes, again highlighting the
dichotomy between lung and blood compartments.
Previous studies examining IFN- production in HIV-infected
individuals have yielded conflicting results. Early studies (20, 22,
23) demonstrated IFN-
production by PBMCs was impaired in patients
with acquired immunodeficiency syndrome (AIDS). However, early in the
course of HIV infection, IFN-
production appears to be preserved
(27). One potential explanation for the decrease in IFN-
production
by PBMCs in subjects with advancing HIV disease is the progressive loss
of CD4+ memory T lymphocytes (30,
37). These cells have been shown to be one of the major producers of
IFN-
(29). Thus differences in the stage of HIV disease progression
in the various study populations likely contributes significantly to
the variability between studies examining PBMC IFN-
production.
Recently, attention has focused on CTLs as an important source of
IFN-, including those found in HIV-infected individuals (10, 15).
Our results show that CD8+ lung
lymphocytes produce and secrete as much IFN-
, if not more, than
CD4+ lung lymphocytes. Because
CD8+ lymphocytes are the
predominant cell found in HIV-infected patients with lymphocytic
alveolitis (1, 12), these cells are clearly an important source of
IFN-
in this setting.
HIV-specific CTLs can be expected to accumulate in areas where there is
increased expression of viral antigens. Although the number of infected
PBMCs is low and exists mainly in a latent stage (21), tissues
containing differentiated macrophages, especially lymphoid tissues,
appear to harbor substantially more infected cells (8, 24, 25). This
may be due to upregulation of viral expression as monocytes
differentiate into tissue macrophages (as seen in other
lentiviruses) or secondary to
enhanced susceptibility of macrophages to de novo HIV infection (11,
26, 31). Regardless of the etiology, increased expression of viral
antigens in various organs can be expected to result in accumulation of
HIV-specific CTLs within that tissue. In this regard,
CD8+ cells actively producing
IFN- have been demonstrated in lymph nodes from HIV-infected
patients (9). It is possible that enhanced production of IFN-
may
occur in any tissue in which HIV-specific CTLs have accumulated in
response to local infection.
As HIV disease progresses, CD8+
CTLs in the alveolar space are replaced by
CD8+CD57+
suppressor cells (16, 28). This switch usually heralds the development
of opportunistic infections and the onset of AIDS. Our data suggest
that this period is associated with a loss of IFN- production by
alveolar lymphocytes. We have direct evidence for this in one patient
who was studied over a 4-yr time span. In 1994, the
patient had a peripheral blood CD4 count of 389 cells/µl and a BAL CD4-to-CD8 cell ratio of 0.12. His
lung lymphocytes secreted large amounts of IFN-
(6,529 pg/ml). Four
years later, his CD4 count is 178 cells/µl and BAL CD4-to-CD8 cell
ratio is 0.14. However, his lung lymphocytes now make no IFN-
.
Whether this phenomenon reflects an actual loss of IFN-
-secreting
cells, inability of suppressor cells to secrete this cytokine, or
inhibition of IFN-
secretion from other sources by
CD8+CD57+
T-cell suppressive factors is not known. The downregulation of IFN-
production does not appear to represent just a Th1-to-Th2 T-cell switch
inasmuch as secretion of IL-4 or IL-10, two Th2 cytokines, was rarely seen.
These studies have important implications on the management of
HIV-related pulmonary diseases. Some investigators (2, 19) have
suggested that IFN- should be given to HIV-infected individuals in
an attempt to augment AM immune function against diseases such as
Pneumocystis carinii pneumonia and
other opportunistic pathogens. Our findings support this hypothesis
because patients with late-stage HIV disease, in whom opportunistic
infections are most common, indeed have an impaired ability to produce
IFN-
locally in the lung. However, attempts to augment pulmonary
immune function through administration of exogenous IFN-
in
HIV-positive individuals with higher CD4 counts may be unnecessary.
Furthermore, one must remain cognizant about the potential for IFN-
to upregulate HIV infection in latently infected mononuclear phagocytes
(3). It is likely that any attempts at pulmonary immunomodulation in HIV-infected patients will have to be performed on an individual basis
after thorough evaluation of the existing infectious and/or immunologic milieu.
In summary, our study demonstrates that lung lymphocytes from
HIV-positive patients before the onset of AIDS produce and secrete substantial amounts of IFN-. These findings have important
implications for understanding the pathogenesis of pulmonary
complications in HIV-infected individuals.
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ACKNOWLEDGEMENTS |
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We thank the staff in the Infectious Disease Research Clinic at Indiana University and at the Damian Center (Indianapolis, IN) who were instrumental in recruiting patients and acquiring clinical data.
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FOOTNOTES |
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This investigation was supported by National Heart, Lung, and Blood Institute Clinical Investigator Development Award HL-02703 and Grant HL-53231; National Center for Research Resources Grant MO1-RR-750; and National Institute of Allergy and Infectious Diseases Grant AI-25859-07 (AIDS Program).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: H. L. Twigg III, Dept. of Medicine, Indiana Univ. Medical Center, 1001 West 10th St., OPW 425, Indianapolis, IN 46202.
Received 2 February 1998; accepted in final form 8 October 1998.
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