Production of interferon-gamma by lung lymphocytes in HIV-infected individuals

Homer L. Twigg III, Blake A. Spain, Diaa M. Soliman, Kenneth Knox, Richard A. Sidner, Carol Schnizlein-Bick, David S. Wilkes, and Gary K. Iwamoto

Divisions of Pulmonary/Critical Care Medicine and Infectious Diseases, Department of Medicine, and Department of Surgery, Indiana University Medical Center, Indianapolis, Indiana 46202


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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-gamma (IFN-gamma ), 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-gamma spontaneously and in response to phytohemagglutinin A. IFN-gamma 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-gamma . However, this ability was lost in patients with late-stage disease. Correlation between blood and lung lymphocyte IFN-gamma secretion was poor, suggesting regional differences in lymphocyte function. These data suggest that lung levels of IFN-gamma are high until late in HIV disease. These findings support the concept of administering exogenous IFN-gamma to patients with late-stage HIV disease and opportunistic infections.

lymphocytic alveolitis; cytotoxic T lymphocytes; suppressor cells; macrophage activation; human immunodeficiency virus


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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-gamma (IFN-gamma ) 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-gamma 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-gamma may be actively secreted by alveolar T cells. In this study, we analyzed IFN-gamma production and secretion by lung and peripheral blood lymphocytes in patients at various stages of HIV infection.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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-gamma , 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-gamma 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-gamma 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-gamma 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, 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-gamma (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-gamma 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-gamma 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.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Secretion of IFN-gamma 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-gamma 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-gamma 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-gamma spontaneously. After stimulation with PHA, lung lymphocytes from both groups secreted significantly more IFN-gamma 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-gamma secretion, suggesting that both T-cell populations were producing IFN-gamma .


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Fig. 1.   Interferon-gamma (IFN-gamma ) secretion by lung lymphocytes from human immunodeficiency virus (HIV)-positive patients (n = 16) and normal volunteers (n = 4). Lung lymphocytes (106 cells/ml) were cultured in complete medium supplemented with 10% human serum (HS) for 48 h in absence [unstimulated (Unstim)] and presence of 1 µg/ml of phytohemagglutinin A (PHA). Supernatants were harvested and analyzed for IFN-gamma with an ELISA. * P < 0.05 vs. Unstim lymphocytes.

In two subjects, IFN-gamma was not secreted by lung or blood lymphocytes. In the remaining individuals, correlation between blood and lung lymphocyte IFN-gamma secretion was poor, suggesting regional differences in lymphocyte function (Fig. 2). In some subjects, PHA-stimulated lung lymphocytes secreted more IFN-gamma 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-gamma than autologous lung lymphocytes.


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Fig. 2.   Comparison of PHA-induced IFN-gamma secretion between blood and lung lymphocytes from HIV-positive patients (n = 14). Lung and blood lymphocytes (106 cells/ml) were cultured in complete medium supplemented with 10% HS for 48 h in presence of 1 µg/ml of PHA. Supernatants were harvested and analyzed for IFN-gamma with an ELISA.

CD4+ lymphocytes have traditionally been thought of as the major producers of IFN-gamma (29), and, indeed, some investigators have found increased IFN-gamma production early in HIV infection before depletion of CD4+ cells (27). Recent studies (10, 15) have shown that CD8+ CTLs also produce IFN-gamma when they encounter target antigens. Thus experiments were performed to determine which lymphocyte subsets were actively secreting IFN-gamma in the lungs and blood of HIV-infected subjects. Purified lymphocyte subsets were isolated by immunomagnetic separation techniques and analyzed for spontaneous IFN-gamma secretion. The results from two experiments shown in Table 1 demonstrate that CD8+ lymphocytes are major producers of IFN-gamma in the lungs and blood of HIV-infected individuals. These experiments demonstrate that lung CD8+ cells secrete as much IFN-gamma , if not more, than CD4+ cells. Furthermore, in these two experiments, lung CD8+ cells tended to secrete more IFN-gamma than blood CD8+ cells, again suggesting a compartmentalized pulmonary response.

                              
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Table 1.   Spontaneous IFN-gamma secretion by lung and blood T-cell subsets in HIV-infected subjects

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-gamma as demonstrated by the 17-kDa band corresponding to the molecular mass of mature IFN-gamma , which may reflect spontaneous cell activation through excessive manipulation or magnetic beads. IFN-gamma secretion was slightly greater when lymphocytes were stimulated with PHA. Secretion of IFN-gamma by CD8+ lung lymphocytes was greater than the corresponding peripheral blood population. Immunoprecipitation studies demonstrated that PHA-stimulated lung lymphocytes actively synthesized IFN-gamma . Furthermore, immunoprecipitation studies on T-cell subsets again demonstrated that IFN-gamma was actively produced by both CD4+ and CD8+ blood and lung lymphocytes (data not shown).


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Fig. 3.   IFN-gamma Western blot of blood and lung lymphocyte subset culture supernatants from an HIV-infected individual with and without PHA stimulation. Supernatant proteins were resolved by SDS-PAGE, electroblotted to nitrocellulose, and probed for IFN-gamma with polyclonal rabbit anti-human IFN-gamma antibody. After incubation with goat anti-rabbit IgG-peroxidase, signal was detected by enhanced chemiluminescence techniques. Nos. at left and right, molecular mass in kDa.

Northern blots. Steady-state IFN-gamma mRNA levels were determined through Northern blot analysis. Figure 4 depicts IFN-gamma 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-gamma than similarly stimulated blood lymphocytes. Interestingly, a faint signal for IFN-gamma message was seen in unstimulated freshly isolated lung lymphocytes but not in blood lymphocytes. The presence of IFN-gamma mRNA in freshly isolated lung lymphocytes suggests there is ongoing production of IFN-gamma in the lungs of HIV-infected individuals.


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Fig. 4.   Northern blot analysis of IFN-gamma gene expression in lung and blood lymphocytes from HIV-positive individual. Whole cell RNA was isolated from freshly harvested lung and blood lymphocytes (-) and from lung and blood lymphocytes that had been cultured at 106 cells/ml for 16 h in 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, transferred to nitrocellulose, and hybridized with a 32P-labeled IFN-gamma probe. After hybridization, blots were washed and exposed to XAR-2 film. kB, kilobases.

Changes in IFN-gamma 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-gamma . Figure 5A shows lung lymphocyte IFN-gamma 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-gamma 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-gamma 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-gamma 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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Fig. 5.   Spontaneous (Unstim) and PHA-induced (PHA) IFN-gamma secretion by lung (A) and blood (B) lymphocytes from HIV-positive patients at different stages of HIV infection. Lymphocytes (106 cells/ml) were cultured in complete medium supplemented with 10% HS for 48 h in absence and presence of 1 µg/ml of PHA. Supernatants were harvested and analyzed for IFN-gamma with an ELISA. CD4 > 500, >500 cells/µl, n = 5 patients; CD4 200-500, 200-500 cells/µl, n = 6 patients; CD4 < 200, <200 cells/µl, n = 5 patients. * P = 0.025 vs. PHA-stimulated lung lymphocytes in CD4 < 200 and CD4 > 500 groups. dagger  P < 0.1 vs. PHA-stimulated blood lymphocytes in CD4 count < 200 group.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

In this study, we investigated secretion of IFN-gamma 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-gamma (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-gamma . The ability to secrete IFN-gamma is associated with increased IFN-gamma mRNA levels and subsequent active protein synthesis. Correlation between blood and lung lymphocyte IFN-gamma is poor, suggesting that regulation of IFN-gamma production differs in the pulmonary and vascular compartments. However, in some individuals, lung lymphocytes secrete more IFN-gamma than autologous blood lymphocytes, suggesting an augmented ability to produce and secrete IFN-gamma at the tissue level. Finally, we demonstrated that the ability of alveolar lymphocytes to secrete IFN-gamma is most pronounced in HIV-infected subjects in the middle stages of their disease. Early in HIV infection, when pulmonary complications are uncommon, IFN-gamma secretion by lung lymphocytes is similar to that in HIV-negative subjects, with elevated lymphocytes in BAL. More importantly, late in HIV infection, IFN-gamma levels decline, providing a theoretical explanation for the increased prevalence of opportunistic infections in late-stage HIV disease. The difference in IFN-gamma 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-gamma production in HIV-infected individuals have yielded conflicting results. Early studies (20, 22, 23) demonstrated IFN-gamma production by PBMCs was impaired in patients with acquired immunodeficiency syndrome (AIDS). However, early in the course of HIV infection, IFN-gamma production appears to be preserved (27). One potential explanation for the decrease in IFN-gamma 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-gamma (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-gamma production.

Recently, attention has focused on CTLs as an important source of IFN-gamma , including those found in HIV-infected individuals (10, 15). Our results show that CD8+ lung lymphocytes produce and secrete as much IFN-gamma , 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-gamma 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-gamma have been demonstrated in lymph nodes from HIV-infected patients (9). It is possible that enhanced production of IFN-gamma 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-gamma 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-gamma (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-gamma . Whether this phenomenon reflects an actual loss of IFN-gamma -secreting cells, inability of suppressor cells to secrete this cytokine, or inhibition of IFN-gamma secretion from other sources by CD8+CD57+ T-cell suppressive factors is not known. The downregulation of IFN-gamma 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-gamma 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-gamma locally in the lung. However, attempts to augment pulmonary immune function through administration of exogenous IFN-gamma in HIV-positive individuals with higher CD4 counts may be unnecessary. Furthermore, one must remain cognizant about the potential for IFN-gamma 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-gamma . These findings have important implications for understanding the pathogenesis of pulmonary complications in HIV-infected individuals.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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