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Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (α-SP-R210n) or nonbinding C-terminal domain (α-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5–10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (α-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-γ secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (α-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-α but increased production of IL-10. Inhibition of IFN-γ production by α-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-β1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-β1.
The function of pulmonary surfactant was originally found to be the reduction of surface tension at the alveolar air-liquid interface, preventing alveolar collapse during respiration. However, recent studies have established that surfactant also regulates pulmonary immune defense against infections and local inflammatory responses . The immunomodulatory functions of surfactant are primarily mediated through surfactant proteins A (SP-A) and SP-D , which belong to the mammalian collectin family of proteins that includes mannose-binding lectin and conglutinin [3, 4]. All collectins have an amino-terminal collagen-like stalk and a carboxy-terminal c-type lectin domain, the latter binding carbohydrate-containing molecules on the cell walls or membranes of infectious agents. Recognition of microorganisms by collectins triggers innate immune responses that facilitate microbial clearance.
SP-A is one of the most abundant SPs and is produced primarily by alveolar type II cells. SP-A enhances innate immunity by increasing phagocytosis of pathogenic microorganisms, including intracellular pathogens such as Mycobacterium tuberculosis [1, 5, 6]. SP-A can also up-regulate the synthesis of reactive oxygen intermediates and secretion of inflammatory cytokines [7, 8]. On the other hand, SP-A mediates resolution of inflammation  through enhanced clearance of apoptotic neutrophils [10, 11], suppression of cytokine production induced by Gram-negative organisms , and inhibition of NADPH oxidase . In addition, studies in SP-A null mice suggested that SP-A regulates adaptive immunity in vivo [9, 14, 15], and corresponding in vitro studies show that SP-A influences migration and differentiation of APC . Furthermore, SP-A suppresses allergen- and mitogen-induced T cell proliferation [15, 17–19] and IL-2 secretion , suggesting that SP-A regulates inflammation through inhibiting cell-mediated immunity. The inhibition of mitogen-induced lymphocyte proliferation in PBMC was associated with binding of SP-A to its receptor SP-R210 .
SP-R210 was originally purified from macrophage cell lines and alveolar type II cells  and subsequently shown to be present on human monocytes and nonadherent cells in PBMC . Although several other mammalian proteins also bind SP-A [22–25], only SP-R210 has been shown to mediate inhibition of T cell proliferation and IL-2 secretion by SP-A . However, the mechanisms underlying this inhibition remain unclear. We recently identified SP-R210 as unconventional myosin 18A (Myo18A) . Alternative splicing of the Myo18A gene produces several long and short isoforms in a tissue- and cell-specific manner. A short isoform, SP-R210, serves as an extracellular receptor for SP-A in macrophages . The antibodies generated against the neck region of the molecule, designated as α-SP-R210n, blocked SP-A binding, whereas the antibodies to the carboxy-terminal domain, α-SP-R210ct, did not, indicating that SP-R210n contains the SP-A-binding site . Based on previous work indicating that SP-A inhibits lymphocyte proliferation through SP-R210 , the present study determined whether SP-R210 regulates lymphocyte proliferation and cytokine production during adaptive recall immune responses to M. tuberculosis antigens.
Polyclonal antibodies against SP-R210n or SP-R210ct were generated as described previously  by immunizing rabbits with recombinant domains of SP-R210 . IgG from preimmune or immune sera was purified by affinity chromatography on a HiTrap protein G-sepharose column (GE Healthcare-Biosciences, Uppsala, Sweden). The purity of the preparation was assessed by SDS-PAGE, and the protein content was measured by the bicinchoninic acid (BCA) colorimetric assay (Pierce, Rockford, IL, USA). The endotoxin concentrations in αSP-R210n, αSP-R210ct, and preimmune IgG were in the range of 3.5–6.3 pg/μg IgG, as determined by the QCL-1000 Limulus amoebocyte lysate assay (Cambrex, Walkersville, MD, USA). Purified antibodies were lyophilized and stored at −70°C until use, and they were dissolved in sterile PBS before use. PE-conjugated antibodies to human CD3 (clone UCHT1) and CD14 (clone 61D3) were purchased from eBioscience (San Diego, CA, USA). FITC-conjugated anti-rabbit IgG was obtained from R&D Systems (Minneapolis, MN, USA) or Molecular Probes (Eugene, OR, USA). Neutralizing antibodies against IL-10 and TGF-β1 were purchased from R&D Systems. Human SP-A was isolated from spent alveolar proteinosis fluid, as described previously .
Heat-killed, whole-washed M. tuberculosis Erdman strain was kindly provided by Dr. Patrick Brennan (Colorado State University, Fort Collins, CO, USA) and used at 2.5 μg/ml as an antigen to stimulate PBMC. We also used 1 μg/ml anti-CD3 (OKT3, a generous gift from Ortho Biotechnology, Raritan, NJ, USA) and 0.5 μg/ml anti-CD28 (BD Biosciences, San Jose, CA, USA) to stimulate CD3+ T cells.
Blood was obtained from healthy tuberculin skin test-positive [purified protein derivative-positive (PPD+)] and -negative (PPD−) donors according to protocols approved by the Institutional Review Board of the University of Texas Health Center (Tyler, TX, USA). PBMC were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare-Biosciences). CD3+ cells were isolated from PBMC by negative selection with the Pan T Cell Isolation Kit II (Miltenyi Biotec Inc., Auburn, CA, USA), with a purity of >95%, as measured by flow cytometry with a FACSCalibur (BD Biosciences).
PBMC were cultured at 2 × 106 cells/ml in RPMI 1640, supplemented with 5 mg/ml glutamine, 100 μM nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated pooled human AB serum (Atlanta Biologicals, Norcross, GA, USA) in a 96-well plate and incubated at 37°C in 5% CO2 for different time-points in the presence of heat-killed M. tuberculosis or medium alone, as indicated. The cell culture supernatants were collected after 48 h incubation, aliquoted, and stored at −70°C until the cytokine concentration was measured by ELISA.
Freshly isolated or cultured PBMC were washed and resuspended in FACS buffer, composed of Dulbecco’s PBS, pH 7.4, containing 2% heat-inactivated goat serum and 0.5% BSA at a cell density of 5–10 × 106 PBMC/ml. The cells were then distributed in 100 μl aliquots in Eppendorf tubes containing 2.5 μg preimmune IgG, α-SP-R210ct, or α-SP-R210n. After 30 min on ice, the cells were washed twice in FACS buffer and incubated for an additional 30 min with predetermined dilutions of FITC-conjugated goat anti-rabbit IgG and PE-conjugated mouse anti-human CD3 or CD14 mAb or rat isotype control IgG. The cells were washed twice in FACS buffer and resuspended in PBS, and the stained cells were analyzed by flow cytometry using a FACSCalibur (BD Biosciences).
The CFSE-based cell proliferation assay was performed as described previously . Briefly, PBMC were washed three times with sterile HBSS, resuspended at 2 × 107 cells/ml in HBSS, and incubated with 2 μM CFSE (Invitrogen Life Technologies, Gaithersburg, MD, USA) at 37°C for 3 min. Labeling was then stopped by adding an equal volume of human serum, and cells were washed three times with serum-free RPMI 1640. The cells were then resuspended in RPMI 1640, supplemented with 10% pooled human AB serum, and cultured under different conditions as indicated. After 96 h, the cells were collected, washed, and resuspended in FACS staining buffer, and the distribution of CFSE-labeled cells was evaluated by flow cytometry.
The levels of IFN-γ, TNF-α, and IL-10 were measured in culture supernatants by sandwich ELISA using commercially available capture and detection antibodies (BD Biosciences).
Total RNA of PBMC cultured under different conditions was extracted with TRIzol LS reagent (Invitrogen Life Technologies), and 250 ng total RNA was treated with DNase I and reverse-transcribed to cDNA, as described previously . Expression of IFN-γ and 18S rRNA was measured by real-time PCR using commercial primers and probe sets (Applied Biosystems, Foster City, CA, USA). Reactions were performed with the ABI Prism 7700 sequence detection system (Applied Biosystems). The expression levels of IFN-γ mRNA were calculated using the Δ comparative threshold (Ct) method after normalization for 18S rRNA and expressed as a ratio of M. tuberculosis-stimulated cells/cells cultured in medium alone: ΔCt = CtIFN-γ − Ct18srRNA; ΔΔCt sample = ΔCtIFN-γ with M. tuberculosis (M. tb) − ΔCtIFN-γ medium; fold change = 2−ΔΔCt sample.
PBMC were collected after incubation with 2.5 μg/ml heat-killed M. tuberculosis for different time-points, total cell protein extracts were prepared, as described previously , and the protein concentration was measured by the BCA method (Pierce), aliquoted, and stored at −70°C until use. SDS-PAGE and Western blotting were performed as described previously , loading 35 μg cell protein extracts for each sample and blotting with α-SP-R210ct or α-SP-R210n. The blots were stripped and reblotted for β-actin (Abcam Inc., Cambridge, MA, USA) as a loading control.
The paired or unpaired Student’s t-test was performed with GraphPad InStat3 software (GraphPad Software, Inc., San Diego, CA, USA), and P < 0.05 was considered statistically significant.
The α-SP-R210ct and α-SP-R210n antibodies were used previously to study the expression of SP-R210 on murine tissues and cells and in human THP-1 and U937 cell lines , but their binding activities and the expression of their target molecule SP-R210 have not been studied in human primary lymphocytes. To study the effect of these antibodies on human adaptive immune responses, we first determined if these antibodies bind specific cellular subpopulations of PBMC.
First, freshly isolated PBMC from three PPD+ donors were stained with α-SP-R210ct, in combination with PE-conjugated antibodies to CD14 or CD3, to identify monocytes and T cells that express SP-R210, respectively. Figure 1, A and B, showed that α-SP-R210ct binds to virtually all CD14+ cells and a small fraction of CD3+ T cells in freshly isolated, unstimulated PBMC. However, the percentage of CD3+ cells that binds with α-SP-R210ct increased five- to eight-fold in 48 h M. tuberculosis-stimulated PBMC (Fig. 1, C and D). Similar results were obtained with antibody to α-SP-R210n (data not shown).
To show that increased antibody binding to M. tuberculosis-stimulated PBMC correlated with elevated expression of their target molecule SP-R210, we performed Western blotting with α-SP-R210ct on whole cell protein extracts from unstimulated and M. tuberculosis-stimulated PBMC from three healthy tuberculin reactors. Figure 2 demonstrates that SP-R210 was markedly induced in M. tuberculosis-stimulated PBMC. The antibodies to SP-R210 detected the expected 210-kDa receptor and another 150-kDa fragment, as shown previously [21, 26, 28]. The latter represents the expression of an alternatively spliced SP-R210 mRNA in murine alveolar macrophages, lymph nodes, and bone marrow cells (Z.C.C. Chroneos, unpublished data). Both SP-R210 species were maximally induced after 24 h stimulation with M. tuberculosis, and this was not a result of differences in sample loading, as evidenced by equal expression of β-actin for all samples.
As SP-A is known to inhibit lymphocyte proliferation [18, 20], we wished to determine the effect of the anti-SP-R210 antibodies on M. tuberculosis-induced proliferation of PBMC from PPD+ persons. Using a CFSE-based assay, we found that stimulation of PBMC with heat-killed M. tuberculosis induced strong lymphocyte proliferation, which was inhibited by α-SP-R210n in a dose-dependent manner but not by α-SP-R210ct or preimmune IgG (Fig. 3A). These results were consistent in five PPD+ persons (Fig. 3B), and 10 μg/ml α-SP-R210n reduced the M. tuberculosis-induced percentage of proliferating cells to 18.9 ± 3.1% from 46.1 ± 7.3% for PBMC cultured with heat-killed M. tuberculosis and 10 μg/ml preimmune IgG (P=0.009). In contrast, α-SP-R210ct at the same concentration did not reduce proliferation (43.5±4.5% vs. 46.1±7.3% for preimmune IgG, P>0.05). These results suggest that α-SP-R210n is an agonistic antibody that mimics the T cell inhibitory effects of SP-A via binding to its receptor on PBMC.
Secretion of IFN-γ is essential for T cell-mediated immunity against M. tuberculosis and other intracellular pathogens. SP-A reduces production of IL-2, IL-4, and IL-5 by lymphocytes [15, 18, 20, 32], but the effects on IFN-γ production are not well-defined. Stimulation of PBMC from 12 PPD+ persons with heat-killed M. tuberculosis increased secretion of IFN-γ from 30 ± 13 pg/ml to 4291 ± 697 pg/ml, as we and others have shown previously . Addition of α-SP-R210n to M. tuberculosis-stimulated PBMC markedly reduced IFN-γ concentrations by 75% to 1081 ± 375 pg/ml (P=0.0005; n=12). In contrast, preimmune IgG and α-SP-R210ct had no effect on M. tuberculosis-induced IFN-γ secretion (Fig. 4A). To evaluate the effects of α-SP-R210n in the absence of APC, purified CD3+ cells from PBMC of three PPD− donors were stimulated with α-CD3 and α-CD28 in the presence or absence of α-SP-R210n. Under these conditions, α-SP-R210n did not reduce IFN-γ production compared with results obtained with preim-mune IgG at the same concentration (7318±1136 pg/ml vs. 7400±1295 pg/ml, P>0.05). This suggests that the inhibitory effect of α-SP-R210 is mediated through APC rather than a direct effect on T cells.
TNF-α is produced by Th1 cells and mononuclear phagocytes in response to M. tuberculosis infection and is essential for effective immunity . As in the case of IFN-γ, stimulation of PBMC from PPD+ donors with M. tuberculosis increased TNF-α concentrations from 36 ± 15 pg/ml to 2739 ± 259 pg/ml. Preimmune IgG and α-SP-R210ct had no major effect on TNF-α production (2242±190 pg/ml and 2275±419 pg/ml, respectively), whereas α-SP-R210n reduced M. tuberculosis-stimulated TNF-α concentrations by almost 70% to 1039 ± 168 pg/ml (P=0.001, Fig. 4B).
As α-SP-R210n inhibited production of the proinflammatory cytokines, IFN-γ and TNF-α, we wished to determine if this was mediated by increased production of the anti-inflammatory cytokine IL-10, which inhibits IFN-γ secretion by Th1 cells . Addition of heat-killed M. tuberculosis induced IL-10 secretion by PBMC from 19 ± 8 pg/ml to 366 ± 60 pg/ml. The presence of α-SP-R210n further increased M. tuberculosis-induced IL-10 nearly four-fold to 1360 ± 143 pg/ml (P<0.0001, n=15, Fig. 4C), whereas preimmune IgG and α-SP-R210ct had no effect (474±78 pg/ml and 381±58 pg/ml, respectively).
To determine if the antibodies used in this study directly induced cytokine secretion by PBMC in the absence of M. tuberculosis stimulation, PBMC from five PPD− donors were cultured with medium alone or with 10 μg/ml preimmune IgG, α-SP-R210n, or α-SP-R210ct for 48 h, and cytokine concentrations were measured in culture supernatants. Mean IFN-γ concentrations were extremely low, ranging from 5 to 18 pg/ml, and did not differ between groups (data not shown). The two antibodies and preimmune IgG elicited production of modest concentrations of TNF-α, and mean values ranged from 300–900 pg/ml (Fig. 5A). This may be a result of binding of rabbit IgG to the FcRI on monocytes, as reported previously . In contrast to the findings with IFN-γ and TNF-α, α-SP-R210n induced production of 1394 ± 287 pg/ml IL-10 compared with 74 ± 42 pg/ml for cells treated with preimmune IgG and 62 ± 32 pg/ml for those treated with α-SP-R210ct (Fig. 5B, P=0.002, compared with α-SP-R210n). These findings suggest that cross-linking of SP-R210 through the SP-A-binding domain with α-SP-R210n directly stimulates PBMC to produce IL-10, which in turn inhibits secretion of M. tuberculosis-stimulated IFN-γ and TNF-α.
To determine if the effects we observed with α-SP-R210n paralleled those of SP-A, we added 5–20 μg/ml purified human SP-A to M. tuberculosis-stimulated PBMC from four PPD+ persons. SP-A inhibited IFN-γ production in a dose-dependent manner, and 20 μg/ml reduced IFN-γ production by 60% (1168±487 pg/ml vs. 2978±2131 pg/ml). This concentration of SP-A also inhibited M. tuberculosis-induced TNF-α secretion by PBMC by 30% (579±111 pg/ml vs. 813±148 pg/ml) and increased M. tuberculosis-stimulated IL-10 secretion by 43% (420±66 pg/ml vs. 241±48 pg/ml). The effects of SP-A on production of IFN-γ, TNF-α, and IL-10 paralleled those of α-SP-R210n, although they were generally more modest. Thus, we concluded that α-SP-R210n behaved as an agonistic antibody.
The results above indicate that α-SP-R210n inhibits production of IFN-γ and stimulates secretion of IL-10. To determine if the reduction in IFN-γ was mediated through IL-10, we added IL-10 neutralizing antibodies to PBMC from five PPD+ persons that were cultured with M. tuberculosis in the presence of α-SP-R210n. Anti-IL-10 partially reversed the inhibitory effect of α-SP-R210n, and IFN-γ concentrations increased from 1323 ± 351 pg/ml to 1963 ± 613 pg/ml (Fig. 6A). As TGF-β1 also inhibits the Th1 response, and SP-A induces alveolar macrophages to produce TGF-β1 , we also tested the effect of neutralizing TGF-β1. Anti-TGF-β1 had effects similar to those of anti-IL-10, increasing IFN-γ levels to 2537 ± 844 pg/ml, and the combination of both antibodies markedly increased IFN-γ concentrations to M. tuberculosis-stimulated levels of 4519 ± 951 pg/ml (P=0.01, n=5, compared with 1323±351 pg/ml with M. tuberculosis and α-SP-R210n only, Fig. 6A).
As IFN-γ expression is regulated primarily at the transcriptional level, we also studied the effect of α-SP-R210n on M. tuberculosis-induced expression of IFN-γ mRNA by PBMC from six PPD+ subjects, using real-time PCR. Addition of α-SP-R210n to M. tuberculosis-stimulated PBMC inhibited IFN-βmRNA expression by more than 90% (39±17-fold, compared with 534±119-fold, P<0.001, Fig. 6B). In contrast, preimmune IgG and α-SP-R120ct had no effect. IFN-γ mRNA levels were increased significantly by anti-IL-10 (244±65-fold vs. 39±17-fold, P=0.004) and α-TGF-β1 (328±77-fold vs. 39±17-fold, P=0.001). When both cytokines were neutralized, IFN-γ mRNA levels reached baseline values of 606 ± 12-fold, similar to those of M. tuberculosis-stimulated PBMC. These findings suggest that α-SP-R210n inhibited M. tuberculosis-induced IFN-γ secretion by PBMC through induction of IL-10 and TGF-β1.
The data presented in the current report demonstrate that anti-SP-R210 antibodies, α-SP-R210n and α-SP-R210ct, bind to more than 90% of CD14+ monocytes and less than 10% of CD3+ cells in freshly isolated PBMC. Stimulation of PBMC from PPD+ donors with heat-killed M. tuberculosis markedly increased the binding of antibodies to CD3+ cells, and this was correlated with increased expression of SP-R210 in PBMC after stimulation. The antibody, α-SP-R210n, inhibited proliferation of lymphocytes and secretion of IFN-γ by PBMC from healthy PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and the antibody α-SP-R210ct had no effect. Inhibition of IFN-γ production depended on the presence of APC. The antibodies against SP-R210n also inhibited M. tuberculosis-induced production of TNF-αand enhanced production of IL-10, and inhibition of IFN-γ production by α-SP-R210n was abrogated by neutralization of IL-10 and TGF-β1 simultaneously. As our studies were performed with polyclonal rather than mAb to SP-R210, we cannot exclude with certainty the possibility that some of the effects observed may be a result of binding of these polyclonal antibodies to unintended targets. Future studies with monoclonal reagents would be helpful to resolve this issue. Nevertheless, our current findings indicate that SP-A, via SP-R210, suppresses cell-mediated, immune responses to M. tuberculosis by enhanced production of IL-10 and TGF-β1 by APC.
Studies in animals and humans, using in vitro and in vivo models, have demonstrated that SPs, especially SP-A, exhibit potent regulatory effects on immunity and inflammatory reactions in the lung [1, 2, 14, 36]. Although the effect of SP-A on innate immunity has been studied extensively [1, 2], its effect on adaptive immunity in humans has been largely limited to studies of cell proliferation and IL-2 secretion in response to mitogens and anti-CD3 mAb [18–20, 32, 37]. In the current report, we evaluated the effects of SP-A in a more physiologically relevant recall immune response to microbial antigen by human primary T cells from healthy persons infected with M. tuberculosis. We found that the antibodies targeted against the SP-A-binding domain (α-SP-R210n) markedly inhibited M. tuberculosis-induced proliferation of lymphocytes and secretion of IFN-γ and TNF-α in a dose-dependent manner (Figs. 3 and and4).4). Anti-SP-R210n also elicited production of high concentrations of the anti-inflammatory cytokine IL-10, and the combination of neutralizing antibodies to IL-10 and TGF-β1 abrogated the inhibitory effects of α-SP-R210n (Fig. 6). Previous studies showed that SP-A induced TGF-β1 production by alveolar macrophages , whereas the effect of SP-A on IL-10 production is more controversial. Chignard and colleagues [38, 39] showed that SP-A inhibited IL-10 production by LPS-stimulated murine alveolar macrophages, and the same group also reported that SP-A stimulated IL-10 production by bone marrow macrophages and U937 cells . In another study, SP-A-mediated uptake of respiratory syncytial virus suppressed virus-induced IL-10 secretion by primary monocytes and U937 monocytic cells . Importantly, SP-A-deficient mice had reduced the Th2 response and IL-10 secretion following pulmonary infection with influenza A virus, compared with wild-type animals , indicating that SP-A regulates secretion of IL-10 in vivo. Underlying these results is the ability of SP-A to interact with pathogens and host cells, acting to block or stimulate pathogen-induced responses on one hand and modulating the inflammatory response of immune cells on the other [1, 2]. Here, direct probing of the functional domain of the SP-A receptor SP-R210 with an antibody revealed that this receptor inhibits Th1 immune responses. The current findings suggest that SP-A binds to SP-R210n, which is present on the vast majority of monocytes, eliciting production of IL-10 and TGF-β1, known to inhibit production of IFN-γ by reducing the capacity of APC to produce IL-12 and to express costimulatory molecules [42, 43]. Our results extend the findings of previous reports that SP-A inhibits expression of co-stimulatory molecules on murine bone marrow-derived dendritic cells and reduces their capacity to support T cell proliferation in response to antigen .
We found that the inhibitory effects of α-SP-R210n on production of M. tuberculosis-induced IFN-γ depended on the presence of monocytes. Although SP-A is primarily produced by airway epithelial cells, many reports document that SP-A is present in multiple tissues, including the gastrointestinal and sinus mucosa, peritoneum, pericardium, and skin [44–47]. SP-A is also present in plasma at low levels, and these levels increase in patients with lung diseases such as pulmonary fibrosis and acute lung injury [48, 49]. We speculate that SP-A plasma levels may be elevated in chronic lung infections such as tuberculosis and that this may contribute to the reduced production of IFN-γ by peripheral blood T cells in response to mycobacterial antigens in tuberculosis patients [33, 50].
The lung is continually exposed to foreign antigens, and uncontrolled inflammatory responses to these antigens could cause significant tissue damage and reduced gas exchange. Alveolar macrophages have potent phagocytic activity but low immunostimulatory capacity and inhibit the response of alveolar T lymphocytes to mitogens, in part, through SP-A [51, 52], which also inhibits the Th2 immune response in vivo in a mouse model of Aspergillus fumigatus-induced allergic disease . We found that expression of SP-R210 was markedly increased in T lymphocytes after stimulation by M. tuberculosis (Fig. 1) and therefore, hypothesize that this may provide a back-up control mechanism to dampen T cell activation, in addition to the immunosuppressive effects of SP-A mediated through alveolar macrophages. Activated T cells that express SP-R210 may become susceptible to the effects of SP-A, which can directly inhibit cell-cycle progression of T cells and attenuate intracellular calcium release in the absence of APC . Further studies are needed to clarify the direct effects of SP-A on T cells.
High levels of TGF-β1 are associated with active pulmonary tuberculosis [50, 53], but its precise role in the pathogenesis of tuberculosis remains unclear. TGF-β1 inhibits expression of SP-A and other SPs by alveolar type II epithelial cells [54, 55], and we recently found that SP-A levels were reduced during the first 15 days of pulmonary Mycobacterium bovis BCG infection, preceding development of adaptive immunity . We speculate that SP-R210 mediates secretion of TGF-β1 by alveolar macrophages, which in turn inhibits SP-A production, facilitating initiation of protective Th1 immune response against tuberculosis. In later stages of infection, SP-R210-mediated TGF-β1 secretion may help control the inflammatory response and reduce tissue damage.
IL-10 is produced during the local immune response to M. tuberculosis [53, 56], but its role in tuberculosis pathogenesis is uncertain. Mice overexpressing IL-10 from T cells had increased susceptibility to reactivation tuberculosis at later stages of infection . On the other hand, IL-10-deficient mice did not have enhanced resistance to tuberculosis . The primary role of IL-10 in the lung is believed to be to reduce excessive inflammation and injury [59, 60]. Alveolar type I and type II epithelial cells, recruited monocytes, and alveolar macrophages are important sources of IL-10 in the lung [60–62], and the latter three cell types are known to express SP-R210 and respond to SP-A [26, 63]. In combination with previous studies, our findings support the hypothesis that SP-R210 is involved in balancing immune responses to mycobacterial infection through secretion of IL-10 and TGF-β1 in vivo.
In conclusion, we found that in a physiologically relevant system, α-SP-R210n antibodies inhibited production of TNF-α and IFN-γ as well as proliferation of antigen-specific T lymphocytes through eliciting enhanced production of IL-10 and TGF-β1. These results provide insight into the receptor-mediated mechanisms that underlie the anti-inflammatory roles of SP-A in innate and adaptive immunity in multiple previous studies. The present experimental approach obviates technical limitations arising from using native or recombinant SP-A molecules that may be contaminated with different amounts of LPS  or TGF-β1 . The agonistic α-SP-R210n antibodies represent a new tool to understand the physiological functions of the SP-A and its receptor SP-R210 in modulating innate and adaptive immune responses.
This work was supported in part by grants from National Institutes of Health (A1063514 to P. F. B. and B. S.; HL068127 to Z. C. C.), the Margaret E. Byers Cain Chair for Tuberculosis Research (P. F. B.), and the Juvenile Diabetes Research Foundation International (5-2007-813 to Z. C. C. and B. S.).