ABSTRACT
Tuberculosis leads to immune activation and increased human immunodeficiency virus type 1 (HIV-1) replication in the lung. However, in vitro models of mycobacterial infection of human macrophages do not fully reproduce these in vivo observations, suggesting that there are additional host factors. Surfactant protein A (SP-A) is an important mediator of innate immunity in the lung. SP-A levels were assayed in the human lung by using bronchoalveolar lavage (BAL). There was a threefold reduction in SP-A levels during tuberculosis only in the radiographically involved lung segments, and the levels returned to normal after 1 month of treatment. The SP-A levels were inversely correlated with the percentage of neutrophils in BAL fluid, suggesting that low SP-A levels were associated with increased inflammation in the lung. Differentiated THP-1 macrophages were used to test the effect of decreasing SP-A levels on immune function. In the absence of infection with Mycobacterium tuberculosis, SP-A at doses ranging from 5 to 0.01 μg/ml inhibited both interleukin-6 (IL-6) production and HIV-1 long terminal repeat (LTR) activity. In macrophages infected with M. tuberculosis, SP-A augmented both IL-6 production and HIV-1 LTR activity. To better understand the effect of SP-A, we measured expression of CAAT/enhancer binding protein beta (C/EBPβ), a transcription factor central to the regulation of IL-6 and the HIV-1 LTR. In macrophages infected with M. tuberculosis, SP-A reduced expression of a dominant negative isoform of C/EBPβ. These data suggest that SP-A has pleiotropic effects even at the low concentrations found in tuberculosis patients. This protein augments inflammation in the presence of infection and inhibits inflammation in uninfected macrophages, protecting uninvolved lung segments from the deleterious effects of inflammation.
Tuberculosis (TB) infects one-third of the world's population, and there are 8 million cases of active TB annually (25). Human immunodeficiency virus (HIV) coinfection accelerates TB infection in patients with reduced numbers of CD4+ cells. In 2000, 9% of new TB cases occurred in AIDS patients (7). Surprisingly, the alveolar macrophage (AM) is the predominant source of HIV type 1 (HIV-1) replication during TB (14, 19). Therefore, understanding the mechanism by which TB affects HIV-1 replication in infected macrophages is important in understanding why TB accelerates the course of AIDS (30, 35).
The host response to Mycobacterium tuberculosis is associated with an increased state of AM activation. This state is characterized by increased production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-1β, tumor necrosis factor alpha (TNF-α), and IL-8 (15, 38). There is a strong correlation between HIV-1 replication and proinflammatory cytokine production in the lungs of patients with TB, raising the possibility that HIV-1 replication and proinflammatory cytokine production are coordinately regulated (18). Like the HIV-1 long terminal repeat (LTR) promoter, the promoters of most cytokines, including IL-1β, IL-6, and TNF-α, contain NF-κB and CAAT/enhancer binding protein beta (C/EBPβ) transcription factor binding sites (1, 12, 18, 24).
C/EBPβ is central to the control of both viral and cytokine promoters in macrophages. There is a dominant negative 16-kDa C/EBPβ which inhibits transcription when its level is 20% of the level of the 37-kDa stimulatory isoform (1, 24). C/EBPβ−/− mice have increased levels of IL-6, suggesting that C/EBPβ is predominantly responsible for repressing IL-6 in vivo (2). It has been shown previously that in the absence of inflammation, inhibitory C/EBPβ is strongly expressed in human AM. However, AM from lung segments infected with M. tuberculosis exhibit loss of the inhibitory isoform which is associated with increased IL-6 levels and HIV viral load (12, 18). However, in vitro, mycobacterial infection of macrophages paradoxically increases production of the inhibitory isoform, suggesting that additional host factors play a role (18).
The predominant route of transmission of M. tuberculosis is via respiratory aerosols, making the alveolar lining fluid and AM the main barriers in the initial host defense. Surfactant proteins are a major component of the alveolar lining fluid, and the most abundant surfactant protein is surfactant protein A (SP-A) (37). SP-A is a hydrophilic protein which is part of the innate immune response in the lung (37). SP-A is a member of the collectin family, which includes SP-D, C1q, and mannose binding proteins, and one of its main functions is facilitation of opsonization of various pathogens by AM (29). Specifically, SP-A has been shown to increase opsonization of M. tuberculosis and Mycobacterium bovis BCG by human macrophages, murine AM, and rat bone marrow-derived macrophages (8, 11, 20, 33).
In addition to its role as an opsonizer, SP-A appears to directly modulate the inflammatory state of macrophages. SP-A can increase or decrease proinflammatory cytokine production by macrophages depending on the source of SP-A, the stimulus, and the type of macrophages treated (21, 26, 28, 34). A recent study by Gardai et al. may resolve these conflicting results (10). In this study, Gardai et al. demonstrated that the globular heads of SP-A are anti-inflammatory. When the head combines with pathogen-associated molecular pattern molecules, the SP-A tail becomes a proinflammatory stimulus.
Recent studies have also demonstrated that SP-A plays an important role in modulation of the host immune response to mycobacteria. SP-A downregulates nitric oxide (NO) production in M. tuberculosis-infected murine AM, and there is a subsequent increase in mycobacterial survival (21). However, conflicting results were obtained with BCG and activated rat bone marrow-derived macrophages. In these studies, SP-A increased both NO and TNF-α production in the setting of mycobacterial infection (34). There are many possible explanations for these paradoxical results, including the species from which the macrophages were derived, the type of mycobacteria, and the use of resting macrophages versus stimulated macrophages. Consequently, the overall role of SP-A in modulating the host inflammatory response to infection with mycobacteria remains unclear.
In humans, SP-A levels are altered by a number of disease states, including bacterial pneumonia, cystic fibrosis, and pulmonary fibrosis (4, 17, 31). Interestingly, patients with three diseases in which there is increased susceptibility to pulmonary mycobacterial infections, HIV, silicosis, and alveolar proteinosis, all have increased levels of SP-A in the bronchoalveolar lavage (BAL) fluid (BALF) (8, 16, 35, 36). However, there is little data on the status of SP-A levels in patients with TB and how these levels change in response to therapy.
Therefore, we analyzed SP-A levels from normal individuals and subjects with TB before antimycobacterial therapy and after 1 month of antimycobacterial therapy. In subjects with TB, the SP-A levels were reduced in the radiographically involved lobes of the lung. We also developed an in vitro model system of mycobacterium-infected macrophages and determined whether declining levels of SP-A were capable of augmenting the ongoing host inflammatory response and HIV-1 promoter activity.
MATERIALS AND METHODS
Clinical studies.This study was approved by the New York University Institutional Review Board, and all subjects gave written informed consent. All normal individuals and subjects with TB underwent bronchoscopy with BAL as previously described (6). BAL was performed with a flexible bronchoscope with local xylocaine anesthesia. BAL was performed on TB patients during the first week of antimycobacterial treatment. Samples were obtained from the radiographically involved and uninvolved segments of the lungs. These subjects then had a second BAL after 1 month of antimycobacterial therapy. The BALF was filtered through sterile gauze to remove mucous and debris and then centrifuged at 400 × g for 10 min to remove cells. The supernatant was divided into aliquots and stored at −70°C till it was analyzed. Cell differentials were obtained by counting 500 random cells. BALF protein levels were determined by a commercially available protein assay performed according to the manufacturer's instructions (Pierce).
SP-A and IL-6 levels and HIV LTR activity.SP-A levels were determined by using a commercially available enzyme-linked immunosorbent assay (ELISA) as described by other workers (13, 27) (Teijin Biomedical, Tokyo Japan). IL-6 levels were determined by using a commercially available high-sensitivity ELISA (R&D Systems, Minneapolis, Minn.) according the manufacturer's instructions. HIV LTR activity was determined by a chloramphenicol acetyltransferase (CAT) ELISA (Roche). All samples were run in duplicate, and the results described below are the means of the two values obtained in each case.
Phospholipid analysis.The total phospholipid composition of BAL samples was quantified by measuring the inorganic phosphate as described by Bartlett (3). Briefly, after phospholipid was extracted with chloroform-methanol as described by Bligh and Dyer (5), samples were incubated in 8.9 N H2SO4 at 210°C for 30 min, after which H2O2 was added to oxidize any remaining carbon. After addition of ammonium molybdonate and ascorbic acid, samples were incubated for 7 min at 100°C, and then samples were measured spectrophotometrically at 820 nm.
In vitro experiments.SP-A isolated from subjects with alveolar proteinosis by differential centrifugation was graciously provided by Dennis Voelker (Denver, Colo.). The endotoxin content was assayed by the Limulus amebocyte assay (BioWhittaker) and was found to be <30 pg/μg of SP-A. In addition, all experiments were performed in the presence of polymyxin B (10 μg/ml; Sigma).
A total of 2 × 106 THP-1 cells (American Type Culture Collection) or THP-1 cells stably transfected with the HIV LTR (BF24) were maintained in RPMI 1640 containing l-glutamine supplemented with penicillin-streptomycin (BioWhittaker) and 10% bovine calf serum (Life Technologies). THP-1 cells were treated with 20 nM 12-O-tetradecanoylphorbol 13-acetate (Sigma, St. Louis, Mo.) and gamma interferon (1 U/ml) for 1 day prior to infection with mycobacteria at a multiplicity of infection of approximately 1 as previously described (32). A BCG or H37Ra stock was filtered through a 5-μm-pore-size filter and vigorously vortexed for 5 min in order to obtain a single-cell suspension prior to infection. For infection assays, SP-A was preincubated with mycobacteria for 60 min prior to infection. Cells and supernatants were collected after 24 h for analysis.
Immunoblotting.Immunoblotting for C/EBPβ was performed as previously described by utilizing a 15% polyacrylamide gel (32). All lanes were normalized for protein content with 30 to 50 μg of protein/lane. The membranes were probed with anti-C/EBPβ (Santa Cruz Biotechnology, Santa Cruz, Calif.). The membranes were developed by using an ECL Plus detection system (Amersham, Piscataway, N.J.). The gels were scanned by laser densitometry (Molecular Dynamics, Sunnyvale, Calif.), and bands were quantitated with Imagequant software (Molecular Dynamics).
Statistics.All numerical data are expressed below as means ± standard errors of the means. P values were derived from a two-tailed Mann-Whitney test by utilizing the Graphpad Prism statistical software (GraphPad, San Diego, Calif.).
RESULTS
SP-A levels are reduced in TB patients and improve with therapy.We performed bronchoscopy on 10 normal volunteers and 11 patients with TB before and 1 month after the institution of antimycobacterial therapy. The groups were matched with respect to sex, HIV status, and age (Table 1). All subjects with TB were acid-fast bacillus culture positive, and 8 of the 11 had a positive sputum smear for acid-fast bacilli on presentation. All subjects had pan-sensitive disease and had a chest radiography (CXR) compatible with pulmonary TB. BALF cell counts in the radiographically involved lobes of subjects with TB showed an increase in the percentage of lymphocytes and neutrophils (polymorphonuclear leukocytes [PMN]) compared to the percentage in the normal individuals and the radiographically uninvolved lobes. This difference persisted 1 month after therapy (Table 2).
Individuals used in this study
Cellular composition of BALF from subjects in the study
SP-A levels were reduced in BALF from involved lobes compared to the levels in BALF from uninvolved lobes (9.3 ± 2.4 versus 25.4 ± 4.2 μg/ml; P = 0.006) and normal individuals (9.3 ± 2.4 versus 22.5 ± 3.3 μg/ml; P = 0.009) (Fig. 1A). There was no significant difference between the uninvolved lobes and the normal individuals. After 1 month of antituberculous chemotherapy all subjects reported improvement in fever and cough. There was a significant increase in the SP-A levels in the involved lobes (9.3 ± 2.4 versus 19.4 ± 3.7 μg/ml; P = 0.001), and there was no change in uninvolved lobes (25.4 ± 4.2 versus 20.9 ± 3.7 μg/ml; not significantly different) (Fig. 1B). In addition, there was no longer a statistical difference in the SP-A levels between the involved lobes and the normal controls. For all groups, there was no significant difference in the total amount of phospholipid in BAL samples (Fig. 1C). Correction of SP-A levels for total protein did not alter the results (data not shown).
SP-A levels were reduced in TB patients and returned to normal after 1 month of antituberculous therapy. BALF from normal individuals and from radiographically involved and uninvolved lung segments of subjects with TB were assayed for SP-A by a commercially available ELISA. (A) SP-A levels were reduced in the involved lung segments of TB subjects compared to the levels in uninvolved lung segments and normal individuals. (B) SP-A levels increased after 1 month of antituberculous therapy. (C) Total phosphorus levels from BALF of all subjects. Data are the means; error bars are SEM.
There was a negative correlation between SP-A levels and the percentage of PMN in all subjects (r = −0.44; P = 0.004) (Fig. 2). A similar correlation was found with the radiographically involved lobes of TB subjects (r = −0.6; P = 0.01). However, there was no correlation between the SP-A levels and the total cell count, the percentage of AM, or the percentage of lymphocytes.
SP-A levels were inversely correlated with the BALF neutrophil count. (A) SP-A levels were inversely correlated with the PMN content in BALF from normal individuals and involved and uninvolved lung segments. (B) SP-A levels were inversely correlated with the involved lung segments of TB subjects.
Low levels of SP-A are capable of potentiating inflammation during in vitro mycobacterial infection.The presence of reduced SP-A levels during TB led us to examine the effects of reducing the levels of SP-A during mycobacterial infection in vitro. THP-1 macrophages infected with mycobacteria exhibited an increase in IL-6 production compared to that of uninfected control macrophages. SP-A at higher doses resulted in a small increase in IL-6 production. Surprisingly, progressively lower doses of SP-A led to further increases in IL-6, and a 1.7-fold increase in IL-6 (5.2 versus 8.9 pg/ml) was observed with 0.01 μg of SP-A per ml (Fig. 3A). In contrast, a similar dose of SP-A resulted in a 50% reduction in IL-6 levels (6.4 versus 4.4 pg/ml) in uninfected controls (Fig. 3A). The differential effects of SP-A in infected and uninfected cells was most significant at a dose of 0.01 μg/ml (P = 0.02).
Low levels of SP-A increased proinflammatory cytokine production in vitro. THP-1 macrophages were infected with either BCG or H37Ra with or without SP-A for 24 h. (A) IL-6 production was increased in infected THP-1 macrophages (M.TB+) at doses as low as 0.01 μg/ml, and the opposite effect was observed in uninfected cells (M.TB−) (n = 5) (B) HIV LTR activity was assayed with a CAT reporter construct. HIV LTR activity was increased in infected THP-1 macrophages at doses as low as 0.01 μg/ml, and the opposite effect was observed in uninfected cells. The asterisk indicates that the P value is 0.02 for a comparison with SP-A-treated uninfected cells.
We repeated these experiments with THP-1 cells stably transfected with the HIV LTR since HIV replication is upregulated in the involved lung segments of subjects that are infected with both HIV and TB. The HIV LTR and numerous proinflammatory cytokines are regulated by similar groups of transcription factors, making the HIV LTR an excellent readout of proinflammatory cytokine gene transcription (14, 18). Similar to IL-6, in the setting of mycobacterial infection, declining doses of SP-A increased HIV LTR activity, and a persistent effect was observed at doses as low as 0.01 μg/ml (Fig. 3B). Conversely, SP-A by itself had an inhibitory effect on HIV LTR activity in uninfected controls (Fig. 3B).
We next sought to determine the mechanism of IL-6 and HIV LTR regulation in this model. Both IL-6 and the HIV LTR are strongly regulated by C/EBPβ. Similar to previously reported data, infection of THP-1 macrophages with mycobacteria increased expression of both the inhibitory and stimulatory isoforms of C/EBPβ (Fig. 4A). SP-A at a high dose (10 μg/ml) had a minimal effect on the absolute levels of C/EBPβ or the ratio of the stimulatory isoform to the inhibitory isoform (S/I ratio). However, with declining doses of SP-A there was loss of the inhibitory isoform of C/EBPβ (Fig. 4A), and there was an overall increase in the S/I ratio. A reduction in the S/I ratio occurred in three independent experiments, and there was a trend toward significance at an SP-A dose of 0.01 μg/ml when densitometry was used for quantification (Fig. 4B) (P = 0.1). Again, this effect was specific for mycobacterial infection as SP-A at a similar dose had no effect on C/EBPβ expression in uninfected cells. These results were confirmed by quantitative densitometry of immunoblots from three separate experiments (Fig. 4B).
Low levels of SP-A increase the C/EBPβS/I ratio. THP-1 macrophages were infected with H37Ra or BCG with or without SP-A for 24 h. (A)Representative immunoblot for C/EBPβ. Infection with mycobacteria increased the levels of both isoforms (compare lanes 1 and 3). SP-A decreased the amount of the inhibitory isoform, and themaximal effect was observed at 0.01 μg/ml (compare lanes 3 and 7). In contrast, a similar dose of SP-A had no effect on C/EBPβ levels in uninfected cells (lanes 1 and 2). M.Tb, M. tuberculosis. (B) Immunoblots from three or four separate experiments were quantified by densitometry. The results are expressed as the fold changesin the S/I ratio relative to non-SP-A-treated controls. SP-A at a dose of 0.01 μg/ml increased the C/EBPβS/I ratio, while SP-A had little effect in uninfected cells. All lanes were normalized for total protein content (50 μg).−M.TB, without M. tuberculosis; +M.TB, with M. tuberculosis.
DISCUSSION
In this clinical investigation we demonstrated that patients with TB have reduced levels of SP-A in their radiographically involved lobes compared to their uninvolved lobes or normal individuals. After 1 month of antimycobacterial chemotherapy, including aerosolized gamma interferon, the SP-A levels returned to normal in the involved lung segments. Importantly, treatment did not alter the SP-A levels of the uninvolved lung segments. Overall, the concentration of SP-A was inversely related to the concentration of PMN in BALF. In vitro, SP-A was capable of increasing production of both IL-6 and the HIV LTR during mycobacterial infection compared to the effects of SP-A by itself. This increase in IL-6 and HIV LTR production correlated with an increase in the S/I ratio of C/EBPβ isoforms, suggesting that this transcription factor is one target of these effects.
The finding that there were reduced levels of SP-A in pulmonary TB patients is consistent with data for other inflammatory diseases of the lung. Baughman et al. described reduced levels of SP-A in patients with bacterial pneumonia (4). Low levels of SP-A have also been found in other inflammatory disorders of the lung, including pulmonary fibrosis and cystic fibrosis (17, 31). Conversely, patients with AIDS-related pneumonia and specifically Pneumocystis carinii pneumonia have elevated levels of SP-A (22, 23). We concluded that the reductions in SP-A levels in TB patients are regional in nature and occur only in the radiographically involved lung segments. This is probably a reflection of the extent of inflammation, as suggested by the negative correlation between SP-A levels and the degree of BAL neutrophilia. Furthermore, the return of SP-A levels to normal after approximately 1 month of successful therapy indicates that these alterations are not permanent. This is in contrast to other chronic disorders of the lungs which are associated with persistently abnormal SP-A levels, including disorders of the lungs of smokers and asymptomatic individuals with HIV (8, 13). However, while this study did include two HIV-infected subjects, the effect of TB was similar to the effect in HIV-negative subjects with TB. Finally, the lack of a change in the total phospholipid content of BALF suggests that the observed changes in SP-A were not due to alterations in the phospholipid concentration.
Some investigators have postulated that increased levels of SP-A are associated with increased susceptibility to TB. This hypothesis is based on studies which showed that there were increased SP-A levels in subjects with silicosis, pulmonary alveolar proteinosis, and HIV, three diseases in which there is increased susceptibility to mycobacterial infection (8, 16, 35, 36). Recent data have extended these observations, and polymorphisms in the SP-A gene are associated with susceptibility to TB in certain populations (9). While the SP-A levels in the uninvolved lobes of TB patients and in treated TB patients were not statistically significantly different than the levels in normal individuals, the small number of subjects in this study does not allow any conclusions to be drawn about SP-A levels and susceptibility to TB.
The significance of low levels of SP-A remains unclear. While many investigators have postulated that low levels of SP-A in BALF are the result of increased inflammation, it is likely that this also alters the inflammatory milieu within the alveolar space. SP-A modulates numerous inflammatory pathways, including NF-κB and NO production, depending on the cell type and stimulus (21, 26, 28, 34). However, studies have yielded conflicting results. Therefore, we sought to determine whether the lower levels of SP-A associated with mycobacterial infection were able to increase the inflammatory response.
We previously documented that AM from normal individuals express high levels of the inhibitory isoform of C/EBPβ (12, 18). However, TB is associated with a loss of this isoform, which increases the S/I ratio. This correlates with increases in the IL-6, IL-8, IL-1β, and TNF-α levels and the HIV load (12, 15, 18). However, initial in vitro models of mycobacterial infection failed to reproduce the in vivo observations, suggesting that additional factors are required. Recent data obtained in our laboratory suggest that addition of activated lymphocytes to macrophages can recapitulate this phenotype (14). Here we describe a similar role for SP-A.
SP-A at doses ranging from 1 to 0.01 μg/ml was capable of increasing IL-6 and HIV LTR activity in macrophages infected with mycobacteria compared to the effects of SP-A alone. One of the targets of this effect is C/EBPβ, a transcription factor which can either stimulate or inhibit inflammatory cytokine promoters depending on which isoform is expressed (1, 24). In the setting of mycobacterial infection, SP-A increased the relative amount of stimulatory C/EBPβ compared to the amount in SP-A-treated uninfected cells, which correlated with the observed increases in IL-6 and HIV-1 LTR activity. Surprisingly, lower concentrations of SP-A were capable of sustaining this increase in the C/EBPβ S/I ratio. This is consistent with the in vivo observation of reduced levels of SP-A in the radiographically involved lobes. Together with the cytokine data, it suggests that the low levels of SP-A generated during TB are capable of further augmenting the inflammatory response, possibly by reducing inhibitory C/EBPβ production. In contrast, SP-A by itself suppressed IL-6 production and HIV LTR activity without altering C/EBPβ expression.
There are many possible explanations for these divergent effects. Other investigators have shown that SP-A is capable of increasing or decreasing inflammatory cytokine production by macrophages infected with mycobacteria (21, 34). Furthermore, a recent study by Gardai et al. provided a potential mechanism for our findings (10). In this study, SP-A mediated its anti-inflammatory effect when its globular head group bound SIRPα and downregulated inflammation. However, once SP-A bound to a pathogen via its globular head, this complex became proinflammatory through binding of the tail to calreticulin/CD91 (10).
In conclusion, we demonstrated that the SP-A levels are reduced during human TB and that the levels return to normal with appropriate therapy. The low levels of SP-A observed during an inflammatory disease of the lungs can significantly augment the host inflammatory response in the setting of mycobacterial infections, maintaining a permissive environment for inflammation.
ACKNOWLEDGMENTS
This work was supported by the Parker B. Francis Foundation, by grants NIH NCRR GCRC MO1 RR00096, NIH RO1 HL57879, and HL59832, and by an ALA career investigator award
FOOTNOTES
- Received 1 July 2002.
- Returned for modification 2 September 2003.
- Accepted 28 October 2003.
Editor: F. C. Fang
- American Society for Microbiology
