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Am J Physiol Lung Cell Mol Physiol 291: L747-L756, 2006. First published April 28, 2006; doi:10.1152/ajplung.00401.2005
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TGF-beta1 in SP-A preparations influence immune suppressive properties of SP-A on human CD4+ T lymphocytes

Steffen Kunzmann,1,4 Jo Rae Wright,2 Wolfram Steinhilber,3 Boris W. Kramer,1 Kurt Blaser,4 Christian P. Speer,1 and Carsten Schmidt-Weber4

1University Children's Hospital, Würzburg, Germany; 2Department of Cell Biology, Duke University Medical Center, Durham, North Carolina; 3Altana Pharma AG, Konstanz, Germany; and 4Swiss Institute of Allergy and Asthma Research, Davos, Switzerland

Submitted 19 September 2005 ; accepted in final form 19 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surfactant protein A (SP-A) and transforming growth factor-beta1 (TGF-beta1) have been shown to modulate the functions of different immune cells and specifically to inhibit T lymphocyte proliferation. The aim of the present study was to elucidate whether the Smad signaling pathway, which is activated by TGF-beta1, also plays a role in SP-A-mediated inhibition of CD4+ T lymphocyte activation. Recombinant human SP-A1 expressed in Chinese hamster ovary cells [rSP-A1m (mammalian)], but not recombinant Baculovirus-derived rSP-A1hyp (hydroxyproline-deficient), suppressed T lymphocyte proliferation and IL-2 mRNA expression. To test whether SP-A induced Smad signaling, a Smad3/4-specific reporter gene was transfected in primary human CD4+ T lymphocytes. Only rSP-A1m, but not rSP-A1hyp, induced Smad-specific reporter genes, Smad2 phosphorylation, and Smad7 mRNA expression. The effect of rSP-A1m was mediated through the TGF-betaRII and could be antagonized by anti-TGF-beta1 neutralizing antibodies and sTGF-betaRII. Western blot and ELISA analysis revealed that rSP-A1m, but not rSP-A1hyp, contained TGF-beta1. TGF-beta1 was responsible for the differences in inhibition of CD4+ T lymphocyte proliferation and activation of the Smad signaling pathway between rSP-A1m and rSP-A1hyp. After acidification, native SP-A, obtained from patients with alveolar proteinosis, also induced Smad signaling in human CD4+ T lymphocytes leading to an increased inhibition of T lymphocyte proliferation, thus indicating the presence of inactive, latent TGF-beta1 in native SP-A samples. Association between SP-A and latent TGF-beta1 provides a possible novel mechanism to regulate TGF-beta1-mediated inflammation and fibrosis reactions in the lung but also leads to possible misinterpretation of immune-modulator functions of SP-A. Monitoring of SP-A preparations for possible TGF-beta1 is essential.

Smad signaling; acidification; surfactant protein A; transforming growth factor-beta1

LYMPHOCYTES IN THE ALVEOLAR compartment are hyporesponsive compared with blood lymphocytes (2) and at least part of this hyporesponsiveness has been attributed to their contact with pulmonary surfactant (43). This hyporesponsive state of alveolar lymphocytes protects the delicate lung tissue from damage and inflammation that would occur if the T lymphocytes were constantly activated in the lung (34). The pathology of asthma and sarcoidosis has therefore been associated with activation of T lymphocytes (31, 34). Several studies indicate that surfactant protein A (SP-A) (44) and transforming growth factor-beta (TGF-beta) (12) participate in the regulation of the expansion of the adaptive immune response by inhibiting the activation of T lymphocytes in the lung.

The biophysical function of surfactant is to reduce surface tension at the alveolar air-liquid interface. However, some of the surfactant components mediate immunological function. SP-A is a 34- to 36-kDa protein and is the major protein component of pulmonary surfactant. It is secreted by the alveolar type II and Clara cells. SP-A was clearly shown to participate in innate immunity, to have opsonic activity, and to modulate function of innate immune cells, such as macrophages and neutrophils (7). The observations were confirmed by studies showing that SP-A knockout mice are more susceptible to bacterial infection (17), viral infections (18), and LPS-induced lung inflammation (4). Previous studies have shown that SP-A inhibited the proliferation of human T lymphocytes (3, 5, 6). In vivo, SP-A seems to be protective against the pathogenesis of allergies (19) and the development of allogenic donor T cell immune response (45). SP-A protects mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens (19). SP-A was also found to inhibit histamine release in the early phase of allergen provocation and to suppress lymphocyte proliferation in the late phase of bronchial inflammation (41). Furthermore, decreased SP-A concentrations were measured in bronchoalveolar lavage fluids of various inflammatory lung diseases, including cystic fibrosis (30), asthma (42), and adult respiratory distress syndrome (13). Recent reports have demonstrated that SP-A is also expressed at extrapulmonary sites, suggesting a possible role of SP-A in the regulation of immune response in organs in addition to the lung (1).

Because SP-A and TGF-beta1 inhibit lymphocyte proliferation, we hypothesized that SP-A may activate the same signaling pathways as TGF-beta1. TGF-beta1 is released by cells as latent, biologically inactive complexes (25). Upon proteolytic activation, TGF-beta1 signals from the cell membrane via TGF-beta receptors to the nucleus by Smad proteins. The aim of the present study was to elucidate whether Smad signaling pathways are involved in suppression of the specific immune system by SP-A and specifically to determine the mechanism by which SP-A inhibits T lymphocyte proliferation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Recombinant (r) human TGF-beta1, anti-IgG1, and sTGF-betaRII were obtained from R&D Systems (Abingdon, UK). Anti-TGF-beta was obtained from BD PharMingen (Heidelberg, Germany) and R&D Systems. rIL-2 was a gift from Dr. C. Heusser (Novartis, Basel, Switzerland). The mouse anti-human CD28 MAb (clone 15E8) was purchased from BD Pharmingen, and the mouse anti-human CD3 MAb (clone CRL 8001) was obtained from American Type Culture Collection (Manassas, VA). Rabbit anti-Smad2-P was a gift from Dr. C.-H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). The pGL3ti (CAGA)12 vector was provided by Dr. S. Itoh (Netherland Cancer Institute, Amsterdam, Netherlands) and the DNTbetaR-II vector by Dr. R. J. Lechleider (National Cancer Institute, Bethesda, MD).

Cloning and expression of recombinant human SP-A1 expressed in mammalian and insect cells. rSP-A1 was expressed and purified as described by García-Verdugo et al. (10). rSP-A1m (mammalian), the recombinant wild-type form of human rSP-A1m (6A2 allele), was expressed in stably transformed Chinese hamster ovary (CHO) cells as described by Voss et al. (37, 38) and was purified from culture supernatant by mannose affinity chromatography. Endotoxin content was 200 pg LPS/mg of SP-A, as determined by limulus amebocyte lysate clotting test (ENDOSAFE, Charles River Laboratories). rSP-A1hyp (hydroxyproline-deficient), the cDNA for human SP-A1 (6A2 allele) contained in the mammalian expression plasmid pMTE HS10/5, was subcloned into the EcoRI site of the Baculovirus expression vector pVL1393. Recombinant Baculovirus stocks were generated as described by the manufacturer (Invitrogen, Breda, The Netherlands). rSP-A1hyp was expressed in SF21 cells with serum-free Insect Express medium (PAA, Marburg, Germany) and purified from the medium by mannose affinity chromatography. The endotoxin content was 56 pg/mg SP-A protein for SP-A1hyp. The biochemical characterizations of rSP-A1m and rSP-A1hyp proteins used in this study have been previously reported (10).

Purification of native SP-A. Native (n) SP-A was purified by mannose affinity chromatography from lavage fluid of patients with alveolar proteinosis as previously described (24). Endotoxin levels in the SP-A preparations were assessed by limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). The nSP-A preparation used in this study contained 600 pg LPS/mg protein. To obtain endotoxin-free nSP-A preparations with high yield, a new purification method was developed (unpublished observations, F. U. Bosch, Altana Pharma, Konstanz, Germany). Briefly, the lavage pellet was washed with buffer 1 (20 mM Tris·Cl, pH 7.5, 5 mM CaCl2, 0.15 M NaCl, 0.1% Triton X-100, 0.02% NaN3) and extracted with a low-salt buffer (5 mM Tris·Cl, pH 8.0, 5 mM sodium EDTA, pH 8.0, 1% Triton X-100, 0.02% NaN3). Insoluble matter was removed by centrifugation. The supernatant was frozen at –20°C and stored overnight, thawed at room temperature, and centrifuged to remove any precipitate. The extract was supplemented with 30 mM octyl-beta-D-glucopyranoside (solid) and loaded on a TMAE Fractogel (Merck) column equilibrated in washing buffer 2 (50 mM Tris·HCl, pH 8.0, 50 mM NaCl, 1% Triton X-100, 0.02% NaN3). The column was washed with buffer 2 and buffer A (20 mM diamino-propane-HCl, pH 10, 0.02% NaNH3). Bound SP-A was eluted with a gradient to 1 M NaCl in buffer A. Pure fractions were pooled and exchanged into 10 mM Tris·HCl, pH 7.5, by diafiltration over a 50-kDa omega membrane (Pall Filtron). The obtained nSP-A protein was 90% pure as judged by SDS-PAGE and contained <5 pg LPS/mg SP-A.

Acidification of SP-A. Acid activation of SP-A proteins was performed by the addition of 1 N HCl to reach pH 2.0 for 1 h at 4°C. The protein solution was then neutralized using 1 N NaOH and used for biological and biochemical analyses. Control medium without SP-A proteins was treated in the same way.

Isolation of CD4+ T lymphocytes. Peripheral blood mononuclear cells were isolated from blood of healthy volunteers by Ficoll (Biochrom, Berlin, Germany) density-gradient centrifugation. The interface cells were washed three times, and CD4+ T lymphocytes were purified using anti-CD4 Dynal magnetic beads and Detach-a-Bead antibodies (both Dynal, Hamburg, Germany). The purity of CD4+ T lymphocytes was initially tested by flow cytometry and was >95%.

Viability detection. Human CD4+ T lymphocytes viability after exposure to rSP-A1 proteins was evaluated by flow cytometry-based quantification of ethidium bromide (1 µM; Sigma Aldrich) uptake as previously described (35).

CD4+ T lymphocyte proliferation assay. CD4+ T lymphocytes were stimulated in 96-well plastic dishes (Costar, Corning, NY), coated with anti-CD3 (1 µg/ml) with or without anti-CD28 (2 µg/ml) in the absence or presence of nSP-A, rSP-A1m, or rSP-A1hyp without or with mouse anti-TGF-beta (R&D Systems), mouse anti-IgG1, sTGF-betaRII, or BSA. Samples in triplicate, containing 2 x 105 T lymphocytes, were incubated for 3 days. Cells were pulsed for 16 h with 1 µCi [3H]thymidine (Hartmann, Braunschweig, Germany) and harvested on glass fiber filters using an automated multisample harvester (LKB, Pharmacia-Wallac, Turku, Finland). Filters were transferred in sample bags with liquid scintillation fluid and analyzed by a beta-scintillation counter (Pharmacia-Wallac). The neutralizing activity of the two approaches (anti-TGF-beta, sTGF-betaRII) was controlled in titrated concentrations. The TGF-beta blocking MAb was compared with the isotype control mouse anti-IgG1, and sTGF-betaRII was compared with BSA.

RT-PCR. Total RNA was isolated from human CD4+ T lymphocytes using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The RNA was eluted in 40 µl water and subjected to reverse transcription; 5 µg total RNA was reverse transcribed by addition of 500 µg/ml oligo(dT)12 primer (Roche, Basel, Switzerland), RNase inhibitor (Roche) (10 U/µl), dNTP (5 mM each dNTP; Qiagen), and Omniscript transcriptase (Qiagen) (0.2 U/ µl) for 1 h at 37°C. The cDNA was denatured at 90°C for 5 min and used for PCR amplification. PCR reactions were performed with Taq polymerase (Qiagen). RT-PCR was performed with the primers listed in Table 1. The primer for TGF-beta1 was used as described before (16). PCR products were loaded next to a standard (1 kb plus, Life Technologies) and analyzed on 1% agarose gels. Image analysis was performed using a fluorescence imager analyzer FLA 3000 (Fuji, Dielsdorf, Switzerland) and quantified with the use of AIDA software (Raytest, Urdorf, Switzerland).


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  		Table 1. Primers used in this study

 
Western blot. Cell lysates (25 µg) or proteins were boiled and loaded next to a molecular-weight marker (Benchmark, Invitrogen) on a NuPAGE 4–12% bis-Tris gel (Invitrogen). The proteins were electroblotted onto a nitrocellulose membrane (Amersham Life Science, Dübendorf, Switzerland); phosphorylated Smad2 was detected with a rabbit anti-Smad2-P antibody, and TGF-beta1 was detected with an anti-TGF-beta1 antibody (BD PharMingen). The blot was visualized with a LAS 1000 camera (Fuji). For estimation of protein quantity, photographs were taken with incremental exposure times. Accumulated signals were analyzed using AIDA software (Raytest).

Transfections and reporter gene assays. The luciferase reporter gene constructs were transiently transfected into primary, human CD4+ T lymphocytes by Nucleofection technology (14). The (CAGA)12-luciferase construct was originally found in the plasminogen activator inhibitor-1, known to be activated after binding of Smad3/4 complexes following TGF-beta1 engagement of the TGF-beta receptor (9). The DNTbetaR-II vector, expressing a truncated form of the type II receptor for TGF-beta (DNTbetaR-II), lacking the cytoplasmic domain, has been previously described (8). CD4+ T lymphocytes were purified as described above and incubated in serum-free AIM-V medium (LifeTechnologies) for 18 h. The pGL3ti (CAGA)12-luciferase reporter gene without or with the DNTbetaR-II expression plasmid was added to 3 x 106 CD4+ T cells, which were previously washed in PBS and resuspended in 100 µl of Nucleofector solution for T cells (Amaxa Biosystems, Cologne, Germany), electroporated using the U-15 program for resting T lymphocytes or the T-22 program for preactivated T lymphocytes of the Nucleofector (Amaxa), and immediately transferred into prewarmed AIM-V medium. Transfected cells were seeded into 24-well plates, and TGF-beta1, nSP-A, rSP-A1m, rSP-A1hyp, anti-TGF-beta, anti-IgG1, sTGF-betaRII, and/or BSA were added to the cells. Twenty-four hours after transfection, luciferase activity in cell lysates was measured by the dual luciferase assay system (Promega Biotech, Madison, WI) according to the manufacturer's instruction in a Berthold Lumat LB 9507 luminometer (Bad Wildbach, Germany). Firefly luciferase activity of (CAGA)12-luciferase was normalized by the activity of Renilla luciferase under the control of the thymidine kinase (TK) promoter of phRL-TK (5 ng). All values were obtained from experiments carried out in triplicate and repeated at least three times. The neutralizing activity of the two approaches (anti-TGF-beta, sTGF-betaRII) was controlled in titrated concentrations. The TGF-beta blocking MAb was compared with the isotype control mouse anti-IgG1, and sTGF-betaRII was compared with BSA.

ELISA. TGF-beta1 was measured by solid-phase sandwich ELISA with a sensitivity of <10 pg/ml (R&D Systems). The ELISA can measure only active TGF-beta1. When indicated, SP-A preparations (50 µl) were acidified as described in Acidification of SP-A. Data shown represent total from three separate experiments.

Statistical analysis. All results shown are representative of three separate experiments. Results are presented as means ± SE. The data were tested for significance using the Student-Newman-Keuls test. P values <0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Differences between the inhibitory effect of rSP-A1m and rSP-A1hyp on human CD4+ T lymphocyte proliferation. To examine the effect of SP-A on T cell proliferation, two different rSP-A1 were used for cell culture experiments, namely, rSP-A1m (human SP-A1 expressed in stably transfected CHO cells) and rSP-A1hyp (produced in the Baculovirus expression system). CD4+ T lymphocytes were stimulated with anti-CD3 MAb or anti-CD3 MAb and anti-CD28 MAb. The addition of rSP-A1m resulted in decreased proliferation of anti-CD3 MAb-stimulated CD4+ T lymphocytes (Fig. 1A) or anti-CD3 MAb plus anti-CD28 MAb (Fig. 1B) in a concentration-related manner. At the highest concentration tested, 50 µg/ml, rSPA-1m inhibited the anti-CD3 MAb-induced proliferation of human CD4+ T lymphocytes to 13 ± 1.7% and the anti-CD3 MAb and anti-CD28 MAb-induced proliferation to 50 ± 2.9%. However, rSP-A1hyp had no effect on CD4+ T lymphocyte proliferation. These data show that rSP-A1m and rSP-A1hyp differ in their capacity to inhibit CD4+ T lymphocyte proliferation.


Figure 1
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  		Fig. 1. A and B: differences between the inhibitory effect of recombinant human surfactant protein-A (SP-A1) expressed in Chinese hamster ovary cells (rSP-A1m) and rSP-A1hyp (hydroxyproline-deficient) on human CD4+ T lymphocyte proliferation. Human CD4+ T lymphocytes were stimulated with anti-CD3 (A) alone or anti-CD3 and anti-CD28 (B) in the absence or in the presence of increasing rSP-A1m or rSP-A1hyp concentrations. The relative incorporation of [3H]thymidine by human CD4+ T lymphocytes is shown. *P < 0.05 compared with mitogen alone. C and D: effect of rSP-A1m on IL-2 production. C: human CD4+ T lymphocytes were stimulated with anti-CD3 in the absence or in the presence of increasing rSP-A1m concentrations for 24 h and RT-PCR for IL-2 and GAPDH was performed. A representative experiment of 3 independent experiments is shown. D: relative incorporation of [3H]thymidine by human CD4+ T lymphocytes stimulated with anti-CD3 MAb and cultured with medium, rSP-A1m or rSP-A1m, and IL-2 for 3 days is shown. *P < 0.05 compared with mitogen alone. #P < 0.05 compared with mitogen and rSP-A1m.

 
To determine whether the observed inhibitory effect of rSP-A1m on anti-CD3 induced proliferation of human CD4+ T lymphocyte is IL-2-dependent, IL-2 mRNA was measured in CD4+ T lymphocytes 24 h after anti-CD3 stimulation with various amounts of rSP-A1m. Recombinant SP-A1m suppressed IL-2 mRNA expression in a concentration-related manner (Fig. 1C). Human CD4+ T lymphocytes showed 50 ± 2.9% lower expression of IL-2 mRNA after addition of 50 µg/ml rSP-A1m compared with control cells. Exogenously added, recombinant IL-2 restored the rate of proliferation of CD4+ T lymphocytes to the level of cells stimulated with anti-CD3 alone (Fig. 1D). These data indicate that rSP-A1m suppressed anti-CD3-induced IL-2 mRNA expression of human CD4+ T lymphocytes and exogenous added IL-2 reversed the inhibitory effect of rSP-A1m on anti-CD3-induced CD4+ T lymphocyte proliferation.

SP-A does not alter cell viability. To determine whether rSP-A1m and rSP-A1hyp altered proliferation of human CD4+ T lymphocytes by induction of cell death, CD4+ T lymphocytes were cultured for 24, 48, and 72 h in the presence of 100 µg rSP-A1m or 100 µg rSP-A1hyp, and viability was assessed with the ethidium bromide exclusion by flow cytometry. No decline in viability compared with AIM-V medium was observed (supplemental material; Fig. E1).1

Potentially suppressive SP-A activates Smad signaling. To analyze the effect of the potentially immunosuppressive rSP-A1m relative to no suppressive rSP-A1hyp, pathway-specific reporter gene constructs were employed to identify relevant signaling pathways. At concentrations up to 100 µg/ml, rSP-A1m, but not rSP-A1hyp, enhanced the reporter gene activity of the (CAGA)12-luciferase construct in a concentration-related manner (Fig. 2A). Recombinant SP-A1m had no influence on the activity of the empty control-vector (pGL3ti; data not shown). To exclude that a possible endotoxin contamination in the rSP-A1m preparation is responsible for the activation on the (CAGA)12-luciferase construct, the effect of LPS on the (CAGA)12-luciferase construct was tested. LPS could not induce the (CAGA)12-luciferase construct (data not shown).


Figure 2
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  		Fig. 2. rSP-A1m, but not rSP-A1hyp, triggers Smad signaling in human CD4+ T lymphocyte. A: luminometric analysis of (CAGA)12-luciferase reporter-transfected human CD4+ T lymphocytes and incubated with AIM-V medium alone, with TGF-beta1, or with increasing concentrations of rSP-A1m or rSP-A1hyp. *P < 0.05 compared with medium alone. B: phosphorylation of endogenous Smad2 protein. Human CD4+ T lymphocytes were stimulated with TGF-beta1, rSP-A1hyp, and rSP-A1m for 1 h. Smad2 phosphorylation was detected by immunoblotting with anti-phospho-Smad2 antibodies. A representative of 3 independent experiments is shown. C: Smad7 mRNA induction by rSP-A1m. Human CD4+ T lymphocytes were stimulated with different concentrations of rSP-A1m or rSP-A1hyp for 2 h in AIM medium. RT-PCR for Smad7 and GAPDH (top) and densitometric analysis of PCR results (bottom) was performed. *P < 0.05 compared with medium alone.

 
Along with Smad3, Smad2 is phosphorylated by TGF-beta1 and Smad2 phosphorylation can be tested as alternative evidence of Smad signaling. Both TGF-beta1 and rSP-A1m, but not rSP-A1hyp, induce Smad2 phosphorylation in human CD4+ T lymphocytes (Fig. 2B).

The Smad7 gene is immediately induced by TGF-beta1 and acts in a negative feedback loop to regulate the intensity or duration of the TGF-beta signal (28). We therefore verified our reporter gene and phosphorylation results using possible Smad7 mRNA regulation by rSP-A1m in human CD4+ T lymphocytes. RT-PCR analysis of Smad7 from human CD4+ T lymphocytes incubated with rSP-A1m revealed that Smad7 mRNA was rapidly induced in response to rSP-A1m, whereas rSP-A1hyp had no effect (Fig. 2C). A 5.8 ± 0.1-fold increase in Smad7 mRNA was induced by 2 h of rSP-A1m stimulation.

Mechanism of Smad signaling activation by rSP-A1m. To clarify whether the effect of rSP-A1m on Smad signaling is mediated by binding of rSP-A1m to the TGF-beta receptor complex on human CD4+ T lymphocytes, CD4+ T lymphocytes were transiently transfected with a truncated form of the type II receptor for TGF-beta (DNTbetaR-II). This construct effectively repressed induction of cotransfected (CAGA)12-luciferase reporter activity not only by TGF-beta1 but also by rSP-A1m (Fig. 3A). These data show that the rSP-A1m induction of Smad signaling in human CD4+ T lymphocytes is dependent on the TGF-beta receptor.


Figure 3
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  		Fig. 3. Characterization of the mechanism of activation of the Smad signaling by rSP-A1m. A: dependence of TGF-betaRII. Human CD4+ T lymphocytes were transfected with the (CAGA)12-luciferase reporter without (TGF-betaRII+/+; solid bars) or with DNTbetaR-II (TGF-betaRII–/–; open bars) and treated with TGF-beta1 or rSP-A1m. *P < 0.05 compared without DNTbetaR-II overexpression. B: dependence of TGF-beta1. Human CD4+ T lymphocytes were transfected with the (CAGA)12-luciferase reporter, treated with TGF-beta1 or rSP-A1m without or with anti-TGF-beta1 and sTGF-betaRII. *P < 0.05 compared with TGF-beta1 or rSP-A1m alone. C: no effect of rSP-A1m on TGF-beta1 mRNA. Human CD4+ T lymphocytes were incubated with rSP-A1m for different time points. RT-PCR for TGF-beta1 and GAPDH was performed (top). Densitometric analysis of PCR results showed in relation to GAPDH expression (bottom). A representative of 3 independent experiments is shown. D: effect of rSP-A1m on TGF-beta1 release. Human CD4+ T lymphocytes were incubated with different concentrations of rSP-A1m for 24 h. The level of TGF-beta1 in cell supernatant was measured by ELISA. *P < 0.05 compared with medium alone. E: detection of TGF-beta1 in rSP-A1m preparations by Western blot. With different concentrations of rTGF-beta1, rSP-A1m and rSP-A1hyp Western blot analyses were done with an anti-TGF-beta1 antibody. A representative experiment of 3 independent experiments is shown. F: detection of TGF-beta1 in rSP-A1m preparation by ELISA. The level of TGF-beta1 in rSP-A1m and rSP-A1hyp preparation was measured by ELISA. *P < 0.05 compared with medium alone.

 
To determine whether the induction of (CAGA)12-luciferase reporter activity by rSP-A1m was caused by TGF-beta1 induction after rSP-A1m treatment, transiently transfected CD4+ T lymphocytes were treated with TGF-beta1 neutralizing antibody or soluble TGF-betaRII-Fc chimeric protein (sTGF-betaRII). Both clearly eliminated activation by exogenous recombinant TGF-beta1, and both also clearly eliminated activation by rSP-A1m (Fig. 3B). Anti-TGF-beta1 and sTGF-betaRII treatment prevented rSP-A1m enhancement of (CAGA)12-luciferase reporter activity, demonstrating that the rSP-A1m induction of Smad signaling is dependent on TGF-beta1.

From these findings, the possibility was tested that rSP-A1m induced TGF-beta1 production, which then acted in an autocrine fashion to activate (CAGA)12-luciferase reporter activity. To assess this possibility, CD4+ T lymphocytes were treated with rSP-A1m, and TGF-beta1 mRNA induction and TGF-beta1 protein expression were measured. Recombinant SP-A1m did not increase TGF-beta1 mRNA in CD4+ T lymphocytes (Fig. 3C). TGF-beta1 could be measured in the supernatant of rSP-A1m-treated human CD4+ T lymphocytes in a concentration-related manner (Fig. 3D). These findings suggest that SP-A may be inducing release of TGF-beta1 from the lymphocytes. However, another explanation for these findings is that rSP-A1m contains TGF-beta1. Therefore, we analyzed by Western blot rSP-A1 for possible TGF-beta1. We could detect TGF-beta1 in rSP-A1m but not in rSP-A1hyp (Fig. 3E). Furthermore, the level of TGF-beta1 in the rSP-A1m and rSP-A1hyp was measured by ELISA, revealing 19.5 ± 0.3 pg TGF-beta1/µg rSP-A1m, whereas no TGF-beta1 could be detected in rSP-A1hyp (Fig. 3F).

TGF-beta1 mediates T cell suppression by rSP-A1m. To determine whether TGF-beta1 is responsible for the observed differences in inhibition of CD4+ T lymphocytes proliferation between rSP-A1m and rSP-A1hyp (Fig. 1, A and B), human CD4+ T lymphocytes were stimulated with anti-CD3. The effect of rSP-A1m in the presence or absence of anti-TGF-beta1 neutralizing antibody and soluble TGF-betaRII-Fc chimeric protein (sTGF-betaRII) was subsequently measured. When CD4+ T lymphocytes were stimulated with anti-CD3, the inhibitory effect of rSP-A1m could be antagonized by anti-TGF-beta1 and sTGF-betaRII (Fig. 4). Incubation with anti-TGF-beta1 antibody and sTGF-betaRII alone had no effect on CD4+ T lymphocyte proliferation. Incubation of rSP-A1m with the isotype control of the anti-TGF-beta antibody mouse anti-IgG1 had no influence on the inhibitory effect of rSP-A1m. These data show that the TGF-beta1 in rSP-A1m was responsible for the observed differences between rSP-A1m and rSP-A1hyp in CD4+ T lymphocyte proliferation.


Figure 4
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  		Fig. 4. TGF-beta1 in rSP-A1m batches is responsible for the differences between rSP-A1m and rSP-A1hyp in inhibition of CD4+ T lymphocyte proliferation. Human CD4+ T lymphocytes were stimulated with anti-CD3 in the absence or in the presence of rSP-A1m without or with anti-TGF-beta, mouse anti-IgG1, or sTGF-betaRII. The relative incorporation of [3H]thymidine by human CD4+ T lymphocytes is shown. *P < 0.05 compared with mitogen and rSP-A1m.

 
Acidification of nSP-A activates latent TGF-beta1 in nSP-A preparations. To determine whether nSP-A preparations, isolated from alveolar proteinosis patients, also contain TGF-beta1, the effect of nSP-A on the activity of the TGF-beta1-sensitive (CAGA)12-luciferase construct was analyzed. nSP-A had no effect on the reporter gene activity at a concentration of 50 µg/ml (Fig. 5A). To test the hypothesis that TGF-beta1 in nSP-A samples could be bound in a latent form, nSP-A samples were acidified (nSP-A*) to activate latent TGF-beta. After acidification, nSP-A* enhanced the reporter gene activity in a concentration-related manner (Fig. 5, A and B) and induced Smad2 phosphorylation (Fig. 5C). This function of nSP-A* could be specifically antagonized by anti-TGF-beta1 and sTGF-betaRII (Fig. 5D). The concentration of active TGF-beta1 in nSP-A samples with and without acidification was measured by ELISA, revealing an 18.5-fold increase of active TGF-beta1 between nonacid-activated nSP-A samples (0.2 ± 0.05 pg TGF-beta1/µg nSP-A) and acid-activated samples (3.7 ± 0.3 pg TGF-beta1/µg nSP-A*) (Fig. 5E). Acid activation of rSP-A1m samples resulted in a 2.5-fold increase in active TGF-beta1 concentration from 19.5 ± 0.5 to 48 ± 1 pg TGF-beta1/µg rSP-A1m, whereas no active TGF-beta1 could be detected in untreated and acid-treated rSP-A1hyp samples (Fig. 5E). nSP-A was purified via two different methods, mannose affinity chromatography and anion-exchange chromatography. Both enhanced the activity of the TGF-beta1 sensitive (CAGA)12-luciferase construct after acidification (Fig. 5, A and F). The concentrations of active TGF-beta1 in the nSP-A samples were in the same range (with mannose affinity chromatography, nonacid activated = 0.2 ± 0.05 pg TGF-beta1/µg nSP-A and acid activated = 3.7 ± 0.3 pg TGF-beta1/µg nSP-A*; with anion-exchange chromatography, nonacid activated = 0.3 ± 0.1 pg TGF-beta1/µg nSP-A and acid-activated = 3.3 ± 0.2 pg TGF-beta1/µg nSP-A*). Together, these results show that acidification increases the ratio between active and latent TGF-beta1 in nSP-A preparations.


Figure 5
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  		Fig. 5. Acidification of nSP-A activated latent TGF-beta1 in nSP-A preparations. A and B: luminometric analysis of (CAGA)12-luciferase reporter-transfected CD4+ T lymphocytes incubated with nonacid-activated nSP-A or acid-activated nSP-A* (A) and increasing concentration of acid activated nSP-A* (B). *P < 0.05 compared with medium alone. C: phosphorylation of endogenous Smad2 protein by acid-activated nSP-A*. Human CD4+ T lymphocytes were incubated with TGF-beta1, nonacid-activated nSP-A, or acid-activated nSP-A* for 1 h. Smad2 phosphorylation was detected by immunoblotting with anti-phospho-Smad2 antibodies. A representative of 3 independent experiments is shown. D: dependence of TGF-beta1. Human CD4+ T lymphocytes were transfected with the (CAGA)12-luciferase reporter, treated with acid-activated nSP-A* without or with anti-TGF-beta1 or sTGF-betaRII. *P < 0.05 compared with nSP-A* alone. E: detection of TGF-beta1 in acid-activated SP-A* of different preparations. The level of TGF-beta1 in nonacid-activated SP-A and acid-activated SP-A* of different SP-A preparations were measured by ELISA. *P < 0.05 compared with medium alone. F: independence of the used purification system for nSP-A. Human CD4+ T lymphocytes were transfected with the (CAGA)12-luciferase reporter, treated with nonacid-activated nSP-A or acid-activated nSP-A*. A purification system for nSP-A anion-exchange chromatography was used. *P < 0.05 compared with medium alone.

 
Acid-activated nSP-A* enhanced suppressive capability of nSP-A on human CD4+ T lymphocyte proliferation. To examine the effect of acid activation of nSP-A on human CD4+ T lymphocyte proliferation, we stimulated CD4+ T lymphocytes with anti-CD3 in the presence of acid-treated and untreated nSP-A. Acid-activated nSP-A* inhibited proliferation to a greater extent than did nonacid-activated nSP-A. Highest concentrations (50 µg/ml) of nonacid-activated nSP-A inhibited the anti-CD3 MAb-induced proliferation by 21 ± 4.7%, whereas acid-activated nSP-A* inhibited proliferation by 60 ± 3.5%. Acid-activated nSP-A* and acid-activated medium had no influence on viability of human CD4+ T lymphocytes (data not shown). The inhibitory effect of nSP-A could be antagonized by sTGF-betaRII (Fig. 6B). These results demonstrate that acidification of nSP-A enhances the capability of nSP-A to inhibit anti-CD3-induced CD4+ T lymphocyte proliferation mediated by TGF-beta1.


Figure 6
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  		Fig. 6. Effect of nonacid-activated nSP-A and acid-activated SP-A* on CD4+ T lymphocyte proliferation. Human CD4+ T lymphocytes were stimulated with anti-CD3 in the absence or in the presence of different concentration of acid or nonacid-activated nSP-A without (A) or with sTGF-betaRII (B). The relative incorporation of [3H]thymidine by human CD4+ T lymphocytes is shown. *P < 0.05 compared with mitogen alone (A). #P < 0.05 compared with mitogen and nSP-A or nSP-A* (B).

 

   DISCUSSION
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The results presented in this study demonstrate that there is a correlation between the levels of active or latent TGF-beta1 in SP-A and their ability to inhibit CD4+ T lymphocyte proliferation. Our findings show that the activation of Smad signaling and inhibition of human CD4+ T lymphocyte proliferation by rSP-A1m produced in CHO cells were due to TGF-beta1. In contrast, SP-A preparations free of TGF-beta1 (Baculovirus-derived rSP-A1hyp) did not activate Smad signaling and did not suppress human CD4+ T lymphocyte proliferation. After acidification, nSP-A* induced Smad signaling and enhanced inhibition of T lymphocyte proliferation, and TGF-beta1 was detectable. Therefore, it appears that latent TGF-beta1 binds to SP-A molecules, which could account for the immunosuppressive effects described for SP-A in vivo and in vitro.

Cross talk between the Smad signaling pathway and other pathways has been described, which can negatively or positively regulate TGF-beta signaling. For example, Smad7 induction by IFN-{gamma} provides a mechanism for transmodulation between the STAT and Smad signal transduction pathways, providing a basis for the known antagonism between TGF-beta and IFN-{gamma} in the regulation of immune cell functions (36). In our system, SP-A could possibly bind directly to a SP-A receptor on human CD4+ T lymphocytes, and this binding would lead to an activation of the Smad signaling. Hepatocyte growth factor signals via such a mechanism and can lead to transient phosphorylation of Smad2 (8). Recent studies showed that SP-A binds to well-known receptors, such as CD14 (33), Toll-like receptor-2 (27), signal inhibitory regulatory protein {alpha}, and calreticulin/CD91 (11), and influences their signaling cascades. However, the data presented here clearly show that the effect of rSP-A1m on Smad signaling is abrogated by overexpression of a truncated form of the type II receptor for TGF-beta and suggests that the effect of SP-A depends on the TGFRII and is not related to a specific SP-A or other receptors. Therefore, SP-A may bind directly to the TGF-beta receptor complex and activates the Smad signaling pathway or SP-A may contain TGF-beta1. The latter possibility is supported by antagonism experiments using anti-TGF-beta1 or sTGFbetaRII that showed that the effect was dependent on TGF-beta1. These data are also consistent with the possibility that rSP-A1m induces the release of TGF-beta1 by human CD4+ T lymphocytes, which then leads to the Smad activation. A similar effect for SP-A was described for alveolar macrophages, in which SP-A enhances apoptotic cell uptake and TGF-beta1 release (32). The present study showed that rSP-A1m did not induce TGF-beta1 mRNA expression in CD4+ T lymphocytes; however, TGF-beta1 could be detected in medium of rSP-A1m-treated human CD4+ T lymphocytes. Further experiments demonstrated that TGF-beta1 could be detected in the rSP-A1m batches. The increase of active TGF-beta1 observed after acid activation of rSP-A1m revealed that also latent TGF-beta1 was present in rSP-A1m preparations; rSP-A1m contained more active than latent TGF-beta1. In contrast, nSP-A contained higher levels of latent TGF-beta1 than active TGF-beta1. Baculovirus-derived rSP-A1hyp contained no active or latent TGF-beta1.

An obvious explanation for the detection of active and latent TGF-beta1 in SP-A samples is that latent TGF-beta1 is copurified with SP-A. nSP-A was obtained from lung lavage, and TGF-beta1 in its latent form can be found in the lung environment. In addition, transfected CHO cells have been reported to secrete latent TGF-beta1 (26). nSP-A prepared by two different methods was tested for TGF-beta1; we reasoned that it was unlikely that TGF-beta1 would copurify with SP-A isolated by these very different methods, unless indeed SP-A bound to TGF-beta1. Our results showed that acid-activated nSP-A* induced Smad signaling independent of the purification method. This finding suggest that TGF-beta1 directly binds to SP-A rather than copurifies with SP-A. SP-A and latent TGF-beta1 are known to have a "high" binding capacity for a variety of ligands. For example, SP-A binds to specific carbohydrate structures present on the surface of multiple bacteria and viruses, which initiates microbial aggregation and facilitates phagocytosis and killing by macrophages, monocytes, and other inflammatory cells (15). Furthermore, SP-A has been shown to bind to particles of pollen grains (20), mite (40), and Aspergillus fumigatus allergens (19). Until now, it has not been known if SP-A can bind to cytokines. Two forms of latent complexes for TGF-beta1 have been described. In the small latent complex, one molecule of mature, active TGF-beta1 is noncovalently associated with one disulfide-bonded propeptide dimer, called latency-associated protein or LAP. In the large latent complex, LAP is linked by disulfide bonds to one member of a family of high-molecular-weight proteins called latent TGF-beta-binding proteins or LTBPs. LTBP associate with the extracellular matrix, permitting storage of TGF-beta (25). Furthermore, LAP is a ligand for the integrin {alpha}vbeta6, which locally regulates TGF-beta1 function (26). Studies are needed to focus on the ability of LTBPs and LAP to bind to SP-A.

nSP-A and rSP-A from different expression systems differ in their state of multimerization or posttranslational modifications (21–23, 29, 38, 39). These differences could be an explanation for the observed differences between the biological activity and/or the level of TGF-beta1. Despite incomplete oligomerization, incomplete glycosylation, and the absence of hydroxyproline, rSP-A expressed in vitro by insect cells provide a model for the study of biological SP-A functions, such as the stimulation of immune cells, binding of carbohydrates, and inhibition of surfactant lipid secretion from isolated type II cells (22). Especially for rSP-A1hyp, which showed no effect on inhibition of T lymphocyte proliferation in our study, a biological activity could be shown by stimulation of TNF-{alpha} release in THP-1 cells (39). Furthermore, it was shown that recombinant rat SP-A produced in CHO cells was physically and functionally similar to native rat SP-A (21). Therefore, differences in the multimeric structure between nSP-A, rSP-A1m, and rSP-A1hyp are not a likely explanation for the observed differences in the biological activity between rSP-A and nSP-A. Because the effects of rSP-A1m and nSP-A could be antagonized by anti-TGF-beta1 neutralizing antibodies and sTGF-betaRII, differences in TGF-beta1 levels are more likely responsible for the relative effects observed.

In view of the present study, further investigation is required to determine the molecular basis of a possible interaction between latent TGF-beta1 and SP-A. The postulated storage function of SP-A for latent TGF-beta1 in the lung would support the proposed model of Gardai et al. (11), which suggests that the local lung milieu influences the biological function of SP-A. In an immunologically quiescent and non-inflamed environment latent (inactive) TGF-beta1 would be bound to SP-A. Inflammation and injury associated changes in the homeostasis of the lung could locally activate latent TGF-beta1, leading to suppression of the inflammation processes activated by the collagenous tail domain of SP-A, as described by Gardai et al. (11). The binding capability of nSP-A for latent TGF-beta1 could be a novel mechanism to regulate TGF-beta1-mediated inflammation and fibrosis reactions in the lung and could represent an additional component of crosstalk between innate and adaptive immunity.

As shown in this study, the amount of active TGF-beta1 and latent TGF-beta1 can differ between SP-A preparations. Different amounts of TGF-beta1 and ratios of active vs. inactive TGF-beta1 may influence the effect of SP-A and could explain, at least in part, the differing reports in the literature about the effect of SP-A on cytokine production.

Together, the presence of active or latent TGF-beta1 in SP-A preparation independently caused by contamination or perhaps by a specific interaction between SP-A and latent TGF-beta1 can lead to misinterpretation of immune-modulator functions of SP-A and illustrates the importance of monitoring SP-A preparations for possible (latent) TGF-beta1.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by the Deutsche Forschungsgemeinschaft (KU 1403/2–1) and National Heart, Lung, and Blood Institute Grant HL-68072 (J. R. Wright), by Swiss National Foundation Grants Nr 31-65436.01 and 32-100266, and 3100A0-100164, the Ehmann Foundation, the Saurer Foundation, Zuerich, and the OPO Foundation, Zuerich.


   ACKNOWLEDGMENTS
 
We thank S. Drewitz and J. Breyer (Altana) for preparation of recombinant SP-As, Kathy Evans for purifying nSP-A, Dr. E. Flory (Paul-Ehrlich Institute) for helpful discussion, and T. Tambuse and B. Ottensmeyer for excellent technical assistant. We are grateful to Dr. S. Itoh (Research Laboratories, Kyowa Hakko Kogyo, Tokyo, Japan) for providing the pGL3ti(CAGA)12 vector, Dr. C.-H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden) for providing the rabbit anti-Smad2-P, and Dr. R. J. Lechleider (National Cancer Institute, Bethesda, MD) for providing the DNTbetaR-II vector.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Kunzmann, Univ. Children's Hospital, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany (e-mail: Kunzmann_s{at}kinderklinik.uni-wuerzburg.de)

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. Section 1734 solely to indicate this fact.

1 The online version of this article contains supplemental data. Back


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