Departments of 1 Medicine and
2 Biochemistry, The Lawson Research Institute,
St. Joseph's Health Center, The University of Western Ontario, London,
Ontario, Canada N6A 4V2; 3 Department of
Pulmonary and Critical Care Medicine, Investigation of
possible mechanisms to describe the hyporesponsiveness of pulmonary
leukocytes has led to the study of pulmonary surfactant and its
constituents as immune suppressive agents. Pulmonary surfactant is a
phospholipid-protein mixture that reduces surface tension in the lung
and prevents collapse of the alveoli. The most abundant protein in this
mixture is a hydrophilic molecule termed surfactant-associated protein
A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human
T lymphocyte proliferation and interleukin-2 production in vitro.
Results presented in this investigation showed that different sources
of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human
T lymphocyte proliferation in a dose-dependent manner. A structurally
similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to
SP-A-treated cultures also did not disrupt inhibition, suggesting that
the effect is not mediated by the carbohydrate recognition domain of
SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid
Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was
responsible for the inhibition of T cell proliferation. A polyclonal
antiserum directed against an SP-A receptor (SP-R210)
completely blocked the inhibition of T cell proliferation by SP-A.
These results emphasize a potential role for SP-A in dampening
lymphocyte responses to exogenous stimuli. The data also provide
further support for the concept that SP-A maintains a balance between
the clearance of inhaled pathogens and protection against collateral
immune-mediated damage.
surfactant-associated protein A; T lymphocyte; proliferation; suppression; receptor
THE HYPORESPONSIVE STATE of pulmonary leukocytes
compared with peripheral blood leukocytes suggests an organ-specific
regulation of immune function (4, 5, 35). It has been suggested that pulmonary surfactant plays a significant role in the induction and
maintenance of this hyporesponsiveness (3, 2, 35, 47). Pulmonary
surfactant is a phospholipid-protein mixture that prevents collapse of
alveoli at the end of expiration. There are four known
surfactant-associated proteins (SPs), termed SP-A, SP-B, SP-C, and
SP-D, which have been isolated from lung lavage. SP-B and SP-C are
hydrophobic molecules, whereas SP-A and SP-D are hydrophilic (32). SP-A
is the most abundant of these proteins, representing 3% of the total
mass of pulmonary surfactant in humans (13). Studies by a variety of
researchers have reported that lymphocyte hyporesponsiveness can be
induced in vitro by the addition of cell-free lung wash (3, 35, 47) or
pulmonary surfactant (2, 47) to stimulated cells. These observations
led to the hypothesis that pulmonary surfactant protects the lung from
immune-mediated damage initiated by inhaled antigens and particles, in
part by influencing pulmonary leukocyte responses (33, 47). We have previously shown that SP-A inhibited T cell proliferation induced by
either phytohemagglutinin (PHA) or anti-CD-3 and also inhibited interleukin-2 (IL-2) secretion from these cells in a dose-dependent manner (7). We also showed that SP-A inhibited T cell proliferation up
to 24 h after the lymphocytes had been treated with mitogen (7).
SP-A belongs to a subgroup of molecules termed collectins (25), which
are composed of a collagen-like region and a carbohydrate recognition
domain (20). More specifically, this molecule is organized into the
following four domains: a short
NH2-terminal segment, a
collagen-like domain possessing reiterated Gly-X-Y triplets, a neck
region that includes a span of hydrophobic amino acids, followed by the
COOH-terminal carbohydrate recognition domain that binds mannose (6).
Examples of other collectins are mannose-binding protein,
conglutinin, CL-43, and SP-D, which differ in their
carbohydrate-binding specificity as well as their oligomeric structure
(20). In the case of SP-A, trimeric subunits associate by the folding
of the collagen-like region into triple helices. Fully assembled SP-A
is composed of six trimeric units that are laterally associated in the
first part of the collagen domain and stabilized by disulfide bridges
at the NH2 terminus. The overall
structural organization is very similar to C1q (10, 12), such that the
molecules appear indistinguishable by electron microscopy (19). It has
also been shown that unlabeled C1q can displace fluorescein
5-isothiocyanate-conjugated SP-A binding to human peripheral blood
monocytes, suggesting that a common receptor(s) may exist on the
surface of these cells (15). Monocytes that were plated on tissue
culture wells coated with C1q or anti-C1qR monoclonal antibody were
less responsive to the opsonic effects of SP-A (15). In contrast, it
has been shown that C1q or soluble C1q receptor only mildly inhibited
SP-A binding to U-937 cells (25). Tenner et al. (40) used human
proteins and demonstrated that C1q only mildly displaced SP-A binding
to rat type II cells and that an anti-C1q antibody that inhibits
C1q-mediated phagocytosis lacked the same activity with SP-A-mediated
phagocytosis. Together, these studies suggest that SP-A and C1q may
share certain common receptors on some cells. SP-A and C1q may also
possess receptors that do not bind these molecules interchangeably. One
such receptor has been characterized on the surface of macrophages and
type II cells and found to bind SP-A even in the presence of a 100-fold excess of mannan (11). Polyclonal antiserum raised against this 210-kDa
receptor (SP-R210) was also able to prevent SP-A from inhibiting
phosphatidylcholine secretion by phorbol ester-treated type II cells
and to inhibit the SP-A-dependent uptake of bacillus Calmette-Guerin by
macrophages (45).
The objective of the current study was to
1) determine if the various sources
and species of SP-A inhibit T cell proliferation to confirm that the
effect is conserved across species and compare these results to a
structurally similar molecule C1q,
2) determine the structural domains
of SP-A that mediate inhibition of T cell proliferation using a panel
of mutant recombinant SP-A proteins, and
3) determine the contribution of a
recently characterized SP-A receptor expressed on leukocytes in
mediating the suppression of
[3H]thymidine
incorporation by T cells.
Isolation of SP-A
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C until used.
Human SP-A. Two preparations of human SP-A were employed in these studies. The first preparation was isolated from the bronchoalveolar lavage of patients with alveolar proteinosis by n-butanol extraction of the sedimented surfactant and further solubilization. Purity and identity were confirmed via Western blot analysis and study of tryptic peptides by tandem mass spectometry (49). The functional activity of SP-A was confirmed by its ability to aggregate lipids and bind mannose (49).
Human SP-A was also prepared from bronchoalveolar lavage obtained from patients with alveolar proteinosis by a method of preparative isoelectric focusing (SP-A IEF; see Ref. 22). After centrifugation of the crude bronchoalveolar lavage, insoluble proteins were recovered by centrifugation, washed with saline, and solubilized in H2O from the cell-free surfactant pellet. SP-A was precipitated with calcium and resolubilized 10 times consecutively to reduce serum protein contamination. The calcium-precipitated pellet was solubilized with mercaptoethanol, urea, and Nonidet P-40 (NP-40) and was used for preparative IEC with a Bio-Rad Rotofor apparatus. The resulting white precipitate that formed was dialyzed for several days against H2O and finally dialyzed against polymixin-agarose to reduce endotoxin to <1.2 pg lipopolysaccharide/µg SP-A (22). This preparation initially exists as large insoluble granules in tissue culture media at room temperature, which we assume becomes more soluble with time at 37°C.
Native rat SP-A. Native rat SP-A was obtained from the bronchoalveolar lavage of adult rats after intratracheal instillation of 40 mg/kg of silica instilled 4 wk before lavage. Butanol extraction of the cell-free bronchoalveolar lavage was undertaken followed by application of the crude protein pellet to an immobilized mannose affinity column in the presence of calcium. After thoroughly washing unbound protein through the column, SP-A was eluted with an EDTA-containing buffer (17).
Recombinant SP-A. Recombinant rat SP-A
was generated using insect cell lines and baculovirus vectors. Briefly,
the 1.6-kb cDNA for rat SP-A (37) was ligated into the pVL 1392 recombination vector, which was then cotransfected into
Spodoptera frugiperda (sf 9)
cells with a modified Autographa californica
virus. The recombinant viruses, generated in situ by homologous
recombination, were then plaque purified and used to infect
Trichoplusia ni cells (29).
Recombinant SP-A was isolated from the serum-free culture media by
affinity chromatography on immobilized mannose. This SP-A has been
shown to have measurable functional properties that are similar to
native SP-A (27). The final preparation was dialyzed against 5 mM Tris
(pH 7.4) and stored at 20°C (27).
Recombinant SP-A mutants. To further specify the region of SP-A affecting T cell proliferation, recombinant SP-As with deletions and point mutations in the major structural domains were developed by site-directed mutagenesis. We used mutagenic oligonucleotides and the polymerase chain reaction to generate telescoping deletions in the cDNA for SP-A by overlap extension (21), as previously described (14). The nucleotides encoding Asn1-Ala7 were deleted, and the signal sequence was directly juxtaposed to downstream sequences encoding Gly8-Phe228. Additional truncated mutant cDNAs were generated in a similar fashion by deletion of the nucleotides encoding Asn1-Gly44 and Asn1-Pro80 and ligation of the native signal sequence to downstream nucleotide sequences encoding Gly45-Phe228 and Ala81-Phe228, respectively. The truncated proteins were denoted N1-A7, N1-G44, and N1-P80. A mutant SP-A was developed in which the collagen-like region was deleted as a cassette, directly joining the intermolecular disulfide-containing NH2-terminal fragment and the neck region of the protein (30). This protein was denoted G8-P80. Finally, mutant SP-As containing mutations that block carbohydrate binding (E195A) or alter carbohydrate-binding specificity (E195Q, R197D) were produced as previously reported (28, 29). The coding region for all mutant cDNAs was sequenced by the dideoxy method of Sanger et al. (36) to confirm the intended deletions and exclude spurious mutations. The production of mutant recombinant SP-A in the baculovirus system was performed as for the wild-type recombinant protein (27, 29).
Cell Isolation and Culture
Lymphocytes and monocytes were obtained from the peripheral blood of healthy volunteers by buoyant density centrifugation using Lymphoprep resolving medium (Nycomed, Oslo, Norway). The peripheral blood mononuclear cells (PBMC) were then washed three times in cold tissue culture media, RPMI-1640 (GIBCO BRL, Burlington, ON, Canada) containing penicillin (100 µg/ml; GIBCO BRL), streptomycin (100 µg/ml; GIBCO BRL), amphotericin B (2.5 µg/ml; GIBCO BRL),T Cell Proliferation Assays
[3H]thymidine incorporation assay was used as a bioassay for lymphocyte function. Two different T cell mitogens were used: 1) PHA-P (Sigma, St. Louis, MO) and 2) anti-CD-3 (UCHT1; ID Laboratories, London, ON, Canada). Several different reagents were used in the T cell proliferation assays, including human C1q (Sigma), different preparations of SP-A, including SP-Ahyp and a panel of mutants, the mannose homopolysaccharide mannan (Sigma), and the rabbit anti-human polyclonal serum directed against a previously reported SP-A receptor (11). Varying amounts of these reagents were added to the stimulated lymphocytes as indicated in RESULTS and in Figs. 1-6 after confirming protein concentrations by the method of Lowry et al. (23). SP-A binds mannose immobilized on affinity chromatography columns. This property was exploited to determine if the carbohydrate portion of SP-A is involved in inhibition of T cell proliferation. In some experiments, mannan was added to cultures at final concentrations of 1 and 5 mg/ml. Cultures were incubated at 37°C with 5% CO2 in a humidified atmosphere for 72 h. At the 60-h time point, 1 µCi/well of [3H]thymidine was added (specific activity 6.7 Ci/mmol; Amersham International, Oakville, ON, Canada). Cells were subsequently harvested with a semiautomated Skatron cell harvester, which bound labeled DNA to glass filter papers via the cell harvester vacuum manifold. Filter papers were dried, and the amount of [3H]thymidine incorporated into DNA was measured via liquid scintillation spectrophotometry. Data are expressed as the means ± SE of the percentage of [3H]thymidine incorporation compared with cultures treated with mitogen only.Western Blot Analysis of PBMC and Nonplastic Adherent PBMC Lysates for Presence of the SP-A Receptor SPR-210
PBMC were isolated as described previously and resuspended at a final concentration of 2 × 106 cells/ml. A 5-ml aliquot was then added to a T-25 flask (Corning) and incubated under standard tissue culture conditions for 90 min with occasional rocking. Nonadherent PBMC were then gently aspirated from the flask and washed two times in serum-free media. Aliquots were added to 1.5-ml Eppendorf tubes (2 × 106 cells/tube) and centrifuged for 10 s in a bench top microfuge. Cell pellets were resuspended in 25 µl of distilled H2O, 12 µl of 5× Laemmli sample buffer, and 25 µl of a lysis buffer containing 1% NP-40 and 2 mM EDTA.To examine whether treating PBMC with the T cell mitogen anti-CD-3 monoclonal antibody for 24 h had an effect on expression of SPR-210, PBMC cultures from three separate donors (5 × 105 cells/well of a 48-well plate) were cultured alone or activated with 50 ng/ml of anti-CD-3 for 24 h under standard culture conditions. Adherent and nonadherent cells were combined with 150 µl of a 5× protease inhibitor cocktail (Sigma) and assayed for protein content before equal protein concentrations of resting and activated cell lysates were resolved by SDS-PAGE. As a negative control, Chinese hamster ovary cells (CHO-K1) were scraped off a culture flask and treated in a similar fashion to resting and activated PBMC. Samples were lysed by freezing and thawing one time, then vortexed and briefly centrifuged before the collection of lysates. Samples were heated at 95°C for 5 min in 5× Laemlli sample buffer containing dithiothreitol before separation on a 7.5% SDS-polyacrylamide gel. Separated proteins were then electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 0.5% instant nonfat milk in Tris-buffered saline (TBS) for 1 h at room temperature and washed three times with TBS. A 1:3,000 dilution of rabbit anti-rat SPR-210 (11) in TBS was then added to the membrane and incubated for 1 h at room temperature on a gently shaking platform. The properties of the anti-SPR-210 antibody have been described in detail previously (11). After three washes with TBS, a secondary goat anti-rabbit IgG-horseradish peroxidase conjugate was diluted 1:20,000 and incubated with the membrane for 1 h followed by three TBS washes. The enhanced chemiluminesence Western blotting detection kit (Amersham Life Sciences) was used to visualize the SPR-210 protein. An EPSON ES-1200C image scanner was used in conjuction with the National Institutes of Health Image computer software to analyze the density of the 210-kDa SPR-210 and its two primary degradation products to quantify differences in the amount of cross-reactive protein detected.
Statistics
All results shown are expressed as the means of a minimum of three separate experiments. Statistical significance of data was determined by a one-way analysis of variance followed by comparison of experimental results between the experimental groups using the Student-Newman-Keuls test. P values < 0.05 were considered significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparison of the In Vitro Inhibitory Action of SP-A From a Variety of Sources
Previously, it was reported that SP-A from alveolar proteinosis patients enhanced concanavalin A-induced proliferation of rat splenocytes (17). To verify our initial observations using the T cell mitogen PHA, we compared SP-A from different species and methods of isolation. In the first of these experiments, native and recombinant (SP-Ahyp) rat SP-As were added in increasing amounts to PBMC activated with PHA-P (1 µg/ml). Figure 1A demonstrates that both preparations can inhibit T cell proliferation in a dose-dependent fashion. Figure 1B shows the results obtained from experiments in which PHA-P (1 µg/ml)-activated PBMC were treated with increasing amounts of bSP-A and two preparations of human alveolar proteinosis SP-A purified via different methods. All three preparations of SP-A inhibited T cell proliferation in a dose-dependent manner. The ability of IEF isolated human SP-A to inhibit T lymphocyte proliferation is consistent with our previous results (7).
|
Evaluation of Which Region of SP-A Inhibits Proliferation of Human PBMC
The effect of mannan, a mannose homopolysaccharide, on the ability of SP-A to inhibit PHA-driven T cell proliferation was tested (Fig. 2). T cell proliferation was compared between cultures treated with bSP-A and mannan with cultures treated with bSP-A alone. No significant difference in [3H]thymidine incorporation was observed at either dose of mannan combined with bSP-A compared with stimulated cultures treated with bSP-A alone.
|
To determine if a structurally similar collagenous protein had the capacity to block the suppressive activity of bSP-A on activated T cells, increasing amounts of C1q were added to PHA (1 µg/ml)-stimulated cells in the presence of 12.5 µg/ml of bSP-A (Fig. 3). The ratios of C1q to SP-A tested (0.16, 0.31, 0.63, 1.25, 2.5, and 5) did not result in any statististical difference from bSP-A-inhibited cultures.
|
To more clearly define the region of SP-A that affects T cell proliferation, recombinant rat SP-As with deletions and point mutations in the major structural domains were developed by site-directed mutagenesis. The proteins expressed in the insect cell system have been shown to have functional activities that are comparable to native SP-A despite incomplete proline hydroxylation (27). Results in Fig. 4 show that native rat SP-A inhibited PHA-stimulated T cell proliferation. The wild-type recombinant SP-A (SP-Ahyp) also inhibited T cell proliferation, albeit with a lower magnitude than native SP-A. Deletion of the first seven amino acids of the globular NH2 terminus (SP-Ahyp,N1-A7) did not greatly alter the inhibitory action of the preparation in vitro nor did deletion of amino acids 1-44 (SP-Ahyp,N1-G44). These proteins have recently been shown to be a mixture of alternatively processed isoforms that form disulfide-dependent dimers via an NH2-terminal cysteine residue (14). SP-A-induced suppression was blocked with two separate deletion mutants shown in Fig. 4. Deletion of amino acids 1-80 (SP-AN1-P80) abolished the in vitro inhibitory activity of SP-Ahyp, but SP-AN1-P80 does not form disulfide-dependent dimers. The loss of inhibitory activity in SP-Ahyp,N1-P80 was not due to loss of intermolecular disulfide bond formation, since SP-Ahyp,G8-P80, which contains the disulfide-forming NH2-terminal segment, was also inactive. In contrast, the mutant SP-A, created by an inactivating E195A with a substitution of Glu195 by Ala, inhibited PHA-stimulated T cell proliferation in a comparable fashion to SP-Ahyp. Similarly, mutant E195Q/R197D with substitutions of Glu195 by Gln and Arg197 by Asp that altered the carbohydrate-binding specificity of the molecule to favor galactose over mannose binding (29) did not result in a disruption of antiproliferative activity. Thus the carbohydrate-binding activity of SP-A is not involved in inhibition of T lymphocyte proliferation, since mutations that inhibit or alter carbohydrate binding do not influence the activity of SP-A in our assays. These data suggest that the domain of SP-A that mediates the inhibition of T cell proliferation is a 36-amino acid portion of the collagen-like domain between amino acids 45 and 80. This region contains several motifs with alternating positive and negative charges including an RGD sequence.
|
Expression of an SP-A Receptor on Human PBMC
Recently, Chroneos et al. (11) characterized an SP-A receptor expressed on the surface of type II pneumocytes, rat bone marrow-derived macrophages, alveolar macrophages, and U-937 cells. Because this receptor was shown not to bind C1q and does not interact with the carbohydrate-binding site of SP-A, it was of interest to determine whether SP-A mediates its inhibitory effect on T cell proliferation through this receptor. Initially, we tested whether human PBMC and monocyte-depleted nonadherent PBMC express this receptor. Shown in Fig. 5A is a Western blot of cell lysates from these two cell populations that shows both cell populations express the 210-kDa receptor. In addition, an ~160-kDa degradation fragment of the receptor was also observed, consistent with previous findings when lysates are not prepared in the presence of protease inhibitors (11).
|
A more defined experiment was then conducted with resting and T cell mitogen-treated PBMC from three different donors (Fig. 5B). After 24 h of culture, adherent and nonadherent cells from both treatment groups were combined with a protease inhibitor cocktail. Equal amounts of protein were resolved in addition to the CHO-K1 control group before transfer and Western blot analysis. Figure 5B shows that cell lysates from T cell mitogen-treated cultures do express more protein that cross-reacts with the anti-SPR-210 antiserum than unstimulated PBMC cultures. This was also determined by use of densitometric analysis of the 210-kDa band and two faster-migrating products of degradation. The mean increase in band densities from T cell mitogen-treated PBMC was over twofold (1.9-, 2.4-, and 4.4-fold increases). Shown in lane 5 of Fig. 5B and confirmed using densitometric analysis is a lack of specific cross-reactivity to CHO-K1 lysates.
Polyclonal Antisera to an SP-A Receptor Blocks the Inhibitory Action of SP-A on Human PBMC
The mechanism of SP-A-mediated inhibition of T cell proliferation was then explored with the same polyclonal antiserum used to detect the SP-A receptor on PBMC. We found that the suppressive action of SP-A on T cell proliferation was blocked by anti-SP-R210 and that treatment of UCHT1-stimulated PBMC with anti-SP-R210 alone resulted in a moderate but significant increase in [3H]thymidine incorporation of anti-CD-3-stimulated PBMC above the amounts observed using the same dilution of normal rabbit serum (Fig. 6).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reviews of the literature have suggested that SP-A and other collectins function as opsonins for defense against pathogens entering the lung (8, 20, 26, 41, 42). The proposed mechanism for this protective role was thought to be via their ability to recognize cell surface carbohydrate residues that distinguish pathogens from the host's own cells. Further evidence for this protective role is that collectins interact with immune cell surface receptors via their collagen-like region and facilitate microbial phagocytosis and intracellular killing of bacteria (20, 25, 38, 43, 47). Other functions of collectins include stimulation of superoxide production as well as chemotaxis (48). These activities constitute an important first line defense for controlling the growth and spread of pathogens before production of significant quantities of neutralizing antibodies. We have previously shown that SP-A reduces the proliferative response of T lymphocytes to mitogens, suggesting that the protein maintains a state of hyporesponsiveness to prevent flooding of the air spaces with inflammatory cells. Our results combined with these previously published antimicrobial studies suggest that SP-A maintains immunological homeostasis in the lung, attenuating both infection and inflammation.
The current study has demonstrated that SP-A from different species and SP-A obtained by different means of isolation also inhibited T cell proliferation. These data indicate that the inhibitory effect is not species specific. Interestingly, preparations of SP-A obtained from patients with alveolar proteinosis were significantly less active in inhibiting T cell proliferation compared with native bSP-A and rat SP-A molecules. There are several potential explanations for this. A significant portion of alveolar proteinosis SP-A is composed of subunits that form nonreducible dimers (44). Under nonreducing conditions, proteinosis SP-A exists as multimers consisting of more than one octadecamer (16). These alterations in the secondary and tertiary structure of the human proteinosis SP-A may render the region of the molecule responsible for in vitro inhibitory activity less accessible to surface receptors on lymphocytes and macrophages. It is also possible that posttranslational or enzymatic modifications of both native and recombinant SP-A may influence the binding affinity of the different SP-A proteins. Species differences may contribute to different binding affinities for the different cell types.
The domains of SP-A that mediate inhibition of T cell proliferation were also explored using mutant recombinant proteins and specific inhibitors. The role of the carbohydrate domain was examined. Mannan did not reverse the inhibitory effect at higher molar concentrations than SP-A. Recombinant rat SP-A containing inactivating mutations (E195A) or substitutions that alter carbohydrate-binding specificity (E195A, R197Q) were as active as SP-Ahyp in inhibition of T cell proliferation. Collectively, these data indicated that inhibition of T cell proliferation by SP-A is not mediated by the carbohydrate recognition domain.
The collagen-like region of SP-A was also studied to determine what effect it may have on the observed antiproliferative effect. We tested whether C1q, a structurally homologous protein to SP-A, could disrupt the suppressive effect of SP-A by coincubation with 12.5 µg/ml of bSP-A and varying amounts of C1q. The results in Fig. 3 demonstrated that molar ratios of C1q to SP-A as high as 5:1 did not significantly reverse SP-A-mediated inhibition of PHA-induced T cell proliferation. An explanation for the appearance of more proliferation at higher concentrations of C1q is most probably due to SP-A binding C1q in vitro, which sterically hinders SP-A-PBMC interactions (31). Of note, in a previous study, we found that the addition of C1q did not result in a dose-dependent increase or decrease in T cell proliferation (9). In these series of experiments, it was assumed that the SP-A and C1q preparations utilized had comparable half-lives in culture. The demonstration that C1q did not interfere with the in vitro action of SP-A on T lymphocyte proliferation at the majority of ratios tested suggests that SP-A may interact with SPR-210, which binds SP-A but not C1q. These results indicate that a property of bSP-A not shared with C1q mediates suppression of T cell proliferation. Consistent with our interpretation, Tenner et al. (40) also showed that a 100-fold excess of C1q (by mass) did not inhibit SP-A binding to type II pneumocytes.
Mutant recombinant SP-As with telescoping deletions from the NH2 terminus were used to find regions that interacted with PBMC. The inhibitory effect of SP-A was retained despite deletions through Ala7 and Gly44 but was lost with deletions of residues through P80. Because the disulfide-dependent assembly of SP-A is partially preserved in N1-A7 and N1-G44, but lost in N1-P80, we evaluated the activity of an additional collagen region deletion, mutant protein G8-P80, which also exists as a monomer (30). This protein is identical to the N1-P80 except that it contains the disulfide bond forming the NH2 terminus of SP-A. The G8-P80 was also inactive as an inhibitor of T cell proliferation, indicating that the sequence of SP-A from G45 to P80 contained an important motif for T cell inactivation. Examination of this region revealed structural domains with alternating positive and negative charges, including an RGD motif at residues 65-67. We cannot however, exclude the possibility that the deletions created changes in the oligomeric structure of SP-A that may also affect inhibition.
Western blot and densitometric analysis of lysates from adherent or nonplastic adherent PBMC as well as from resting and T cell mitogen-treated PBMC suggest that this protein is expressed on lymphocytes in addition to macrophages. Furthermore, these data suggest that T cells may increase the expression of SPR-210 upon activation. Together, the Western blots and results obtained with anti-SPR-210 antiserum in vitro suggest that the antiserum blocked the inhibitory activity of bSP-A by disrupting an SP-A-receptor interaction. Further binding studies with radiolabeled SP-A, isolated PBMC, and the F(ab')2 of the anti-SPR-210 antibody will clarify this interpretation.
A number of receptors expressed on PBMC that recognize the RGD sequence
provide costimulatory signals to activate T cells and macrophages (1,
18, 34). More specifically, it has been shown that immobilized
anti-CD-3 antibody and fibronectin act synergistically to stimulate
CD-4 cell proliferation and that this proliferative effect can be
inhibited with antibody directed against the common -subunit of the
very late antigen subfamily of integrins (26). Cardarelli et al. (10)
also demonstrated that fibronectin increased IL-2 receptor (CD-25)
expression on CD-3-activated PBMC in a synergistic fashion. It is
therefore possible that SP-A inhibits T cell proliferation through
sequences that block costimulatory signals.
In summary, this study shows that SP-A inhibited T cell proliferation in vitro and suggests that this effect is mediated by a specific receptor, which is not recognized by C1q. The domain responsible is a segment of the collagen-like domain, perhaps an RGD motif. The carbohydrate recognition domain does not play a role. These data support the hypothesis that SP-A contributes to the inhibition of in vivo T cell proliferation and that this effect helps maintain the overall hyporesponsive state of pulmonary leukocytes.
![]() |
ACKNOWLEDGEMENTS |
---|
The second preparation of human surfactant protein A isolated by isoelectric focusing was a kind gift of Dr. David Phelps (Pennsylvania State University, Hershey, Pennsylvania).
![]() |
FOOTNOTES |
---|
This research was supported by the Ontario Lung Association/Thoracic Society and the Medical Research Council of Canada (MT 10555) and by an American Lung Association Career Investigator Grant (F. X. McCormack) and National Heart, Lung, and Blood Institute Grant HL-51134 (J. R. Wright).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P. Borron, Dept. of Cell Biology, Box 3709, Duke Univ. Medical Center, Durham, NC 27710.
Received 14 January 1998; accepted in final form 22 May 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alon, R.,
R. Hershoviz,
E. A. Bayer,
M. Wilchek,
and
O. Lider.
Streptavidin blocks immune reactions mediated by fibronectin-VLA-5 recognition through an Arg-Gly-Asp mimicking site.
Eur. J. Immunol.
23:
893-898,
1993[Medline].
2.
Ansfield, M. J.,
and
B. J. Benson.
Identification of the immunosuppressive components of canine pulmonary surface active material.
J. Immunol.
125:
1093-1098,
1980
3.
Ansfield, M. J.,
H. B. Kaltreider,
B. J. Benson,
and
J. L. Caldwell.
Immunosuppressive activity of canine pulmonary surface active material.
J. Immunol.
122:
1062-1066,
1979[Medline].
4.
Ansfield, M. L.,
H. B. Kaltreider,
B. J. Benson,
and
M. R. Shalaby.
Canine surface active material and pulmonary lymphocyte function: studies with mixed lymphocyte culture.
Exp. Lung Res.
1:
3-11,
1980[Medline].
5.
Ansfield, M. J.,
H. B. Kaltreider,
J. L. Caldwell,
and
F. N. Herskowitz.
Hyporesponsiveness of canine bronchoalveolar lymphocytes to mitogens: inhibition of lymphocyte proliferation by alveolar macrophages.
J. Immunol.
122:
542-548,
1979[Medline].
6.
Benson, B.,
S. Hawgood,
J. Schilling,
J. Clements,
D. Damm,
B. Cordell,
and
R. T. White.
Structure of canine pulmonary surfactant apoprotein: cDNA and complete amino acid sequence.
Proc. Natl. Acad. Sci. USA
82:
6379-6383,
1985[Abstract].
7.
Borron, P.,
R. A. W. Velhuizen,
J. F. Lewis,
F. Possmayer,
A. Caveney,
K. Inchley,
R. G. McFadden,
and
L. J. Fraher.
Surfactant associated protein-A inhibits human lymphocyte proliferation and IL-2 production.
Am. J. Respir. Cell Mol. Biol.
15:
115-121,
1996[Abstract].
8.
Brown-Augsburger, P.,
K. Hartshorn,
D. Chang,
K. Rust,
C. Fliszer,
H. G. Welgus,
and
E. C. Crouch.
Site directed mutagenesis of cys-15 and cys-20 of pulmonary surfactant protein D.
J. Biol. Chem.
271:
13724-13730,
1996
9.
Borron, P. J., E. C. Crouch, J. F. Lewis, J. R. Wright, F. Possmayer, and L. J. Fraher. Recombinant rat surfactant-associated
protein-D inhibits human T-lymphocyte proliferation and interleukin-2
production. J. Immunol. In press.
10.
Cardarelli, P. M.,
S. Yamagata,
W. Scholz,
M. A. Moscinski,
and
E. L. Morgan.
Fibronectin augments anti-CD-3-mediated IL-2 receptor (CD25) expression on human peripheral blood lymphocytes.
Cell. Immunol.
135:
105-117,
1991[Medline].
11.
Chroneos, Z. C.,
R. Abdolrasulinia,
J. A. Whitsett,
W. R. Rice,
and
V. L. Shepherd.
Purification of a cell-surface receptor for surfactant protein-A.
J. Biol. Chem.
271:
16375-16383,
1996
12.
Cockshutt, A. M.,
J. Weitz,
and
F. Possmayer.
Pulmonary surfactant-associated protein A enhances the surface activity of lipid extract surfactant and reverses inhibition by blood proteins in vitro.
Biochemistry
29:
8424-8429,
1990[Medline].
13.
Doyle, I. R.,
H. A. Barr,
and
T. E. Nicholas.
Distribution of surfactant protein A in rat lung.
Am. J. Respir. Cell Mol. Biol.
11:
405-415,
1994[Abstract].
14.
Elhalwagi, B. M.,
M. Damodarasamy,
and
F. X. McCormack.
Alternate amino terminal processing of surfactant protein A produces results in cysteinyl isoform required for oligomeric assembly.
Biochemistry
36:
7018-7025,
1997[Medline].
15.
Geertsma, M. F.,
P. H. Nibbering,
H. P. Haagsman,
M. R. Daha,
and
R. Van Furth.
Binding of surfactant protein A to C1q receptors mediates phagocytosis of Staphylococcus aureus by monocytes.
Am. J. Physiol.
267 (Lung Cell Mol. Physiol. 11):
L578-L584,
1994
16.
Hattori, A.,
Y. Kuroki,
T. Katoh,
H. Takahashi,
H. Q. Shen,
Y. Suzuki,
and
T. Akino.
Surfactant protein A accumulating in the alveoli of patients with pulmonary alveolar proteinosis: oligomeric structure and interation with lipids.
Am. J. Respir. Cell Mol. Biol.
14:
608-619,
1996[Abstract].
17.
Hawgood, S.,
B. J. Benson,
and
R. L. J. Hamilton.
Effects of a surfactant associated protein and calcium ions on the structure and surface activity of lung surfactant lipids (Abstract).
Biochemistry
24:
184,
1985[Medline].
18.
Hershoviz, R.,
D. Gilat,
S. Miron,
Y. A. Mekori,
D. Aderka,
D. Wallach,
I. Vlodavsky,
I. R. Cohen,
and
O. Lider.
Extracellular matrix induces tumour necrosis factor-alpha secretion by an interaction between resting rat CD4+ T cells and macrophages.
Immunology
78:
50-57,
1993[Medline].
19.
Holmskov, U.,
S. B. Laursen,
R. Malhotra,
H. Wiedemann,
R. Timpl,
G. R. Stuart,
L. Tornoe,
P. S. Madsen,
K. B. M. Reid,
and
J. C. Jensenius.
Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins.
Biochem. J.
305:
889-896,
1995[Medline].
20.
Holmskov, U.,
R. Malhotra,
R. B. Sim,
and
J. C. Jensenius.
Collectins: collagenous C-type lectins of the innate immune defense system.
Immunol. Today
15:
67-73,
1994[Medline].
21.
Horton, R. M.,
H. D. Hunt,
S. N. Ho,
J. K. Pullen,
and
L. R. Pease.
Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.
Gene
77:
61-68,
1989[Medline].
22.
Kremlev, S. G.,
T. M. Umstead,
and
D. S. Phelps.
Effects of surfactant protein A and surfactant lipids on lymphocyte proliferation in vitro.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L357-L364,
1994
23.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
24.
Luckow, V. A.,
and
M. D. Summers.
Trends in the development of baculovirus expression vectors.
Biotechnology
6:
47-55,
1988.
25.
Malhotra, R.,
J. Haurum,
S. Thiel,
and
R. B. Sim.
Interaction of C1q receptor with lung surfactant protein A.
Eur. J. Immunol.
22:
1437-1445,
1992[Medline].
26.
Matsuyama, T.,
A. Yamada,
J. Kay,
K. M. Yamada,
S. K. Akiyama,
S. F. Schlossman,
and
D. Morimoto.
Activation of CD4 cells by fibronectin and anti-CD-3 antibody: a synergistic effect mediated by the VLA-5 fibronectin receptor complex.
J. Exp. Med.
170:
1133-1148,
1989[Abstract].
27.
McCormack, F. X.,
H. M. Calvert,
P. A. Watson,
D. L. Smith,
R. J. Mason,
and
D. R. Voelker.
The structure and function of surfactant protein Ahydroxyproline and carbohydrate-deficient mutant proteins.
J. Biol. Chem.
269:
5833-5841,
1994
28.
McCormack, F. X.,
A. L. Festa,
R. P. Andrews,
M. Linke,
and
P. D. Walzer.
The carbohydrate recognition domain of surfactant protein A mediates binding to the major surface glycoprotein of P. carinii.
Biochemistry
36:
8092-8099,
1997[Medline].
29.
McCormack, F. X.,
Y. Kuroki,
J. J. Stewart,
R. J. Mason,
and
D. R. Voelker.
Surfactant protein A residues Glu195 and Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion and the facilitated uptake of phospholipid by type II cells.
J. Biol. Chem.
269:
29801-29807,
1994
30.
McCormack, F. X.,
S. Pattanajituilai,
J. J. Stewart,
F. Possmayer,
K. Inchley,
and
D. R. Voelker.
The Cys6 intermolecular disulfide bond and the collagen-like region of rat SP-A play critical roles in interactions with alveolar type II cells and surfactant lipids.
J. Biol. Chem.
272:
27971-27979,
1997
31.
Oosting, R. S.,
and
J. R. Wright.
Characterization of the surfactant protein A receptor: cell and ligand specificity.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L165-L172,
1994
32.
Possmayer, F.
A proposed nomenclature for pulmonary surfactant-associated proteins.
Am. Rev. Respir. Dis.
138:
990-998,
1988[Medline].
33.
Richman, P. S.,
S. Batcher,
and
A. Catanzaro.
Pulmonary surfactant suppresses the immune lung injury response to inhaled antigen in guinea pigs.
J. Lab. Clin. Med.
116:
18-26,
1990[Medline].
34.
Roberts, K.,
W. M. Yokoyama,
P. J. Kehn,
and
E. M. Shevach.
The vitronectin receptor serves as an accessory molecule for the activation of a subset of gamma/delta T cells.
J. Exp. Med.
173:
231-240,
1991[Abstract].
35.
Robinson, B. W. S.,
P. Pinkston,
and
R. G. Crystal.
Natural killer cells are present in the normal human lung but are functionally impotent.
J. Clin. Invest.
74:
942-950,
1984[Medline].
36.
Sanger, F.,
S. Nicklen,
and
A. R. Coulson.
DNA sequencing with chain terminating inhibitors (Abstract).
Proc. Natl. Acad. Sci. USA
74:
5463,
1997.
37.
Sano, K.,
J. Fisher,
R. J. Mason,
Y. Kuroki,
J. Schilling,
B. Benson,
and
D. Voelker.
Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein A (PSP-A).
Biochem. Biophys. Res. Commun.
144:
367-374,
1987[Medline].
38.
Sastry, K.,
and
R. A. Ezekowitz.
Collectins: pattern recognition molecules involved in first line host defense.
Curr. Opin. Immunol.
5:
59-66,
1993[Medline].
39.
Snyder, J. M.,
and
C. R. Mendelson.
Induction and characterization of the major surfactant apoprotein during rabbit fetal lung development.
Biophys. Acta
920:
226-236,
1987[Medline].
40.
Tenner, A. J.,
S. L. Robinson,
J. Borchelt,
and
J. R. Wright.
Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis.
J. Biol. Chem.
264:
13923-13928,
1989
41.
Van Golde, L. M. G. Potential role of surfactant
proteins A and D in innate lung defense against pathogens.
Biol. Neonate 7, Suppl. 1: 2-17, 1995.
42.
Van Iwaarden, J. F.
Surfactant and the pulmonary defense system.
In: Pulmonary Surfactant: From Molecular Biology to Clinical Practice, edited by B. Robertson,
L. M. G. Van Golde,
and J. J. Batenburg. New York: Elsevier, 1992, p. 215-227.
43.
Van Iwaarden, J. F.,
H. Shimizu,
P. H. M. Van Golde,
D. R. Voelker,
and
L. M. G. Van Golde.
Rat surfactant protein D enhances the production of oxygen radicals by alveolar macrophages.
Biochem. J.
286:
5-8,
1992[Medline].
44.
Voss, T.,
K. P. Schafer,
P. F. Nielsen,
A. Schafer,
C. Maier,
E. Hannappel,
J. Maaben,
B. Landis,
K. Klemm,
and
M. Przybylski.
Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung.
Biochim. Biophys. Acta
1138:
261-267,
1992[Medline].
45.
Weikert, L. F.,
K. Edwards,
Z. C. Chroneos,
C. Hager,
L. Hoffman,
and
V. L. Shepherd.
SP-A enhances uptake of bacillus Calmette-Guérin by macrophages through a specific SP-A receptor.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L989-L995,
1997
46.
Weissbach, S.,
A. Neuendank,
M. Pettersson,
T. Schaberg,
and
U. Pison.
Surfactant protein A modulates release of reactive oxygen species from alveolar macrophages.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L660-L666,
1994
47.
Wilsher, M. L.,
D. A. Hughes,
and
P. L. Haslam.
Immunoregulatory properties of pulmonary surfactant: effect of lung lining fluid on proliferation of human blood lymphocytes.
Thorax
43:
354-359,
1988[Abstract].
48.
Wright, J. R.,
and
D. C. Youmans.
Pulmonary surfactant protein A stimulates chemotaxis of alveolar macrophage.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L338-L344,
1993
49.
Zhu, S.,
I. Y. Haddad,
and
S. Matalon.
Nitration of surfactant protein A (SP-A) tyrosine residues results in decreased mannose binding ability.
Arch. Biochem. Biophys.
333:
282-290,
1996[Medline].