INTRODUCTION

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Pulmonary surfactant is a highly surface-active complex of phospholipids and proteins located at the air-liquid interface inside the alveoli. So far, its major role has been considered to be the stabilization of alveolar walls and prevention of alveoli collapse (1). However, surfactant may have an important role in the small airways as well. Studies in vitro have shown that surfactant deficiency may cause small cylindrical airways to collapse (2, 3). Apart from surface activity, several immunomodulatory actions of surfactant have been reported in the literature (4, 5). Richman and colleagues (6) found in laboratory animals that surfactant suppresses the immune lung injury response to inhaled antigen. It has been shown that aerosolized surfactant treatment results in significant improvement in lung function (7). Recently, Kurashima and colleagues (8, 9) reported in adult asthmatics that nebulization of surfactant resulted in dramatic improvement of vital capacity (VC), FEV₁, and drastic changes in surfactant activity of sputum during an acute asthma attack. Moreover, it has also been shown that concentrations of surfactant protein A in bronchoalveolar lavage fluids was lower in asthma patients than in normal subjects (10), suggesting that the role of airway surfactant is more complex than previously thought.

Bronchial asthma has been recognized as a chronic inflammatory disorder of the airways, manifesting increased bronchial responsiveness and reversible airway narrowing in response to a wide range of stimuli (11). The mechanisms of sensitization by airborne allergens such as dust mites and pollen grain, and the development of early-type hypersensitivity into a late-phase airway inflammation are largely undefined. Supposedly, the allergic airway inflammation is triggered by inhalant allergens which cause immediate hypersensitivity reaction by prompting mast cells to release histamine and other mediators. These mediators sustain the reaction into late-phase airway inflammation by activating T lymphocytes to generate cytokines, such as interleukin-4 (IL-4), IL-5, and tumor necrosis factor alpha (TNF-) (12). IL-4- and TNF-- induced leukocyte recruitment, through the upregulation of cell adhesion molecules, may result in an aggravating bronchial inflammation that leads to edema and leakage of plasma proteins into airway spaces (13). It is not known whether surfactant proteins actually participate in the allergic inflammatory processes that are frequently seen in asthmatic patients.

There is increasing evidence suggesting that lung surfactant proteins A (SP-A) and D (SP-D) play a protective role in the complicated pulmonary defense against pathogens (14). SP-A and SP-D are members of the family of C-type lectins known as the collectins, which are involved in the innate immunity of the lung (15). The amino-terminal end of each collectin polypeptide chain consists of a short noncollagenous region, followed by a region of collagen-like sequence composed of repeating Gly-Xaa-Yaa triplets. The carboxyl-terminal portion of the sequence is characterized by a globular structure, known as the carbohydrate recognition domain (CRD), which mediates calcium-dependent binding to carbohydrate on the surface of microorganisms (16, 17). The collagenous regions of three polypeptide chains assemble to form a collagen-like triple helix, with three C-type lectin domains at its C-terminal end (14). Six of these triple-helical subunits make up the overall structure of SP-A, while SP-D is composed of a cruciform-like structure with four arms of equal length (14). SP-A is able to bind via its CRDs to the surface of pathogens. The binding appears to enhance recognition of the pathogens by the alveolar macrophages, leading to increased phagocytosis and respiratory burst activity (18, 19). Human SP-D also binds in a calcium-dependent manner to carbohydrate structures on gram-negative bacteria, fungi, and viruses, bringing about their agglutination (20).

With respect to allergic diseases, Malhotra and coworkers (23) reported that SP-A binds to water-extractable, allergenic glycoproteins from pollen grains in a calcium-dependent manner, suggesting that the interaction of surfactant proteins with certain allergens may have some role in immediate-type hypersensitivity. Previously, we have demonstrated that SP-A and SP-D inhibited the binding of allergen-specific IgE to house dust mite extracts and concluded that these lung surfactant proteins might well be involved in allergen sensitization or the development of allergic reaction (24).

This study was designed to seek answers to these questions. We examined the effect of surfactant proteins on allergen- induced histamine release during the early-phase allergic reactions, and lymphocyte proliferation during the late-phase inflammatory reactions in children with stable asthma, asthmatic children during acute attacks, and compared this with age-matched control subjects.

	METHODS

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Subjects

Subjects consisted of nine asthmatic children with acute asthma attacks (mean age 11.2 yr; 6 boys, and 3 girls) who were admitted to the Emergency Department of National Cheng Kung University Medical Center within 12 h (10.6 ± 1.2 h, mean ± SEM) of the onset of the attack, 21 asthmatic children without symptoms for at least 2 wk (mean age 10.5 yr; 14 boys and 7 girls), and seven age-matched control subjects who had no history of allergy and asthmatic attacks. All subjects were studied with their parent's or guardian's informed consent, and the entire study was approved by the Human Ethics Committee of the hospital. All asthmatic children had a history of atopy and were sensitive to house dust mite (Dermatophagoides pteronyssinus, Der p) allergens. Subjects taking steroids or inhalant -adrenergic therapy before admission were excluded.

Reagents

Lyophilized house dust mite (Der p) was purchased from Allergon (Engelholm, Sweden). The crude mite preparation was extracted with ether. After dialysis with deionized water, the mite extract was lyophilized and stored at 20° C. The mite antigen was dissolved in pyrogen-free isotonic saline (YF Chemical, Taipei, Taiwan) and filtered through 0.2-µm filter (Microgen, Laguna Hills, CA) before use. The T-cell mitogen, phytohemagglutinin (PHA), was purchased from Murex Diagnostics (Dartford, UK).

Preparation of SP-A and SP-D, and of a Recombinant Fragment of SP-D Composed of the Neck Region and Carbohydrate Recognition Domain (N/CRD)

Native human SP-A and SP-D were purified by affinity chromatography from bronchoalveolar lavage fluid obtained from alveolar proteinosis patients by modified methods previously described (25, 26). Patients' lungs were lavaged at 1-wk intervals for therapeutic purposes, using a heat exchanger and a modified perfusion apparatus to improve efficiency. Bronchoalveolar lavage fluid was adjusted to 10 mM with respect to EDTA and centrifuged at 2,000 × g to remove aggregates. The supernatant was recalcified by the addition of 35 mM CaCl₂, and its pH adjusted to 7.4. Maltose agarose (Sigma, St. Louis, MO) was packed into a column, washed, and then equilibrated with buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM CaCl₂, and 0.02% (wt/vol) sodium azide. The SP-A- and SP-D-enriched supernatant was applied to the maltose agarose column. The column was washed with equilibrating buffer containing 1 M NaCl, and then SP-D was eluted with 20 mM Tris-HCl (pH 7.4)-100 mM MnCl₂, whereas SP-A was eluted with an EDTA gradient (10 to 50 mM). Final purification was achieved by gel filtration on a Superose 12 column. Both preparations were judged to be pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Coomassie blue) and Western blotting. They were also checked for contaminating human immunoglobulin G (IgG), IgM, and IgE antibodies by ELISA with anti-human IgG, anti-human IgM, and anti-human IgE-peroxidase conjugates. No immunoglobulin contamination was ever detected. Routine Limulus lysate assay (Limulus Amebocyte Lysate Test; Sigma Chemical) of our purified SP-A, SP-D, and recombinant SP-D (N/CRD) revealed none of the above preparations was contaminated with detectable endotoxin. The anti-human SP-A and SP-D antibodies were raised in rabbits against purified human SP-A and SP-D, respectively. These antibodies were specific for SP-D and SP-A at the working dilution, as assessed by ELISA and Western blotting. A recombinant polypeptide, comprising the neck region and CRD (N/CRD) of human SP-D was expressed in Escherichia coli and isolated in the form of a self- associating homotrimer (17) as previously described.

Cell Isolation and Culture

Human peripheral blood was obtained from study subjects by venipuncture and collected in 10-ml sodium heparin-containing vacuum tubes (Becton-Dickinson, Rutherford, NJ). Mononuclear cells were isolated on a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). The cells were washed three times in cold tissue culture medium (RPMI-1640; Gibco BRL, Grand Island, NY) containing: penicillin (100 µg/ml; Gibco BRL), streptomycin (100 µg/ml; Gibco BRL), and gentamycin (0.1 µg/ml, Gibco BRL). A sample of suspension was incubated on a Petri dish for 60 min at 37° C in 5% CO₂, and nonadherent cells were collected and resuspended at 2 × 10⁶ cells/ml in RPMI 1640 supplemented with 10% (vol/vol) newborn calf serum (Gibco BRL) or autologous human plasma. Cells were cultured at a concentration of 2 × 10⁵ cells per well in flat-bottomed, 96-well sterile plates (Corning, Inc., Corning, NY).

Cell Proliferation Assay

PHA and Der p extract were used as mitogens for lymphocyte proliferation assay. A varying amount of SP-A, SP-D, and SP-D (N/CRD) was added to the stimulated lymphocytes in the tissue culture medium with or without sugar [(100 mM mannose for SP-A, and 100 mM maltose for SP-D and SP-D (N/CRD)]. Glucose was also included as the control. Cultures were incubated at 37° C in a humidified atmosphere with 5% CO₂ for 72 h. At the 68-h time point, each well was pulsed with 1 µCi of [³H]-thymidine (Amersham International, Oakville, ON, Canada) for 4 h at 37° C and then harvested on a PHD cell harvester (Cambridge Technology, Cambridge, MA). [³H]Thymidine incorporation was determined by liquid scintillation counting (Beckman Instruments, New York, NY). After the 72-h incubation, viability was also assessed by trypan blue exclusion and routinely found to be 95 ± 3%. Background counts for wells containing cells that had not been exposed to PHA and Der p were always less than 800 cpm. Results are presented as mean ± SE for the triplicate cultures. The percentage of surfactant protein inhibition was defined as the percentage of (cpm of mitogen-stimulated peripheral blood mononuclear cells [PBMC] in the presence of surfactant protein  cpm of mitogen-stimulated PBMC)/ cpm of mitogen-stimulated PBMC.

Flow-Cytometry Analysis of PBMC Incubated with Surfactant Proteins

PBMC were incubated with various concentrations of SP-D(N/CRD) at 37° C in a humidified atmosphere with 5% CO₂ for 12 h. Cells were then washed and centrifuged at 400 g for 5 min at room temperature and resuspended in Hanks' balanced salt solution (HBSS) before analysis. The aliquots were stained with fluorescein isothiocyanate (FITC)-conjugated anti-human CD11b or human leukocyte antigen DR (HLA-DR) monoclonal antibodies (Immunotech, Marseilles, France). Cells were excited with an argon laser at 488 nm and emission was read at 522 nm with a long-pass filter, and analyzed on a Becton Dickinson FACScan using Lysis II software (Becton Dickinson, Oxnard, CA). Mean fluorescence intensity (MFI) from 5,000 gated monocytes was determined. The relative binding of monocyte surface molecular (CD11b or HLA-DR) is expressed as the ratio of the staining content incubated with surfactant proteins to the basal staining content.

Histamine Release in the Whole Blood of Asthmatic Children in the Presence of SP-A and SP-D

One milliliter of whole blood was collected from each of the sensitized asthmatic patients or control subjects in a heparinized tube (10 U/ml). To assay the total histamine, which was performed with a histamine immunoassay kit (Immunotech, Marseilles, France), 50 µl of the whole blood collected were diluted to 1,000 µl with distilled water (1:20 dilution) and subjected to freezing and thawing twice. The resulting lysed cell suspension was acylated. The remaining blood sample was diluted 1:7 with histamine release buffer. Fifty microliters of each allergen preparation (0.625 to 20 µg of dust mite extract Der p) with or without purified SP-A and SP-D were then added to the wells of round-bottomed microtiter plates, and incubated for 30 min at 37° C. One hundred microliters of diluted blood (1:7) were added to each well and the covered plate was incubated for 30 min at 37° C. The plate was centrifuged for 5 min at 900 × g at 4° C. Then 100 µl samples of supernatant fluid were collected without disturbing the cell pellet and acylated. Next, 50-µl volumes of the various concentrations of acylated histamine standards or test samples (5 µl diluted to 50 µl with distilled water), and 200 µl of enzyme conjugate were added to each well of the ELISA plate which had been precoated with an antihistamine monoclonal antibody. The plate was incubated for 18 h at 4° C. The plate was then washed three times in order to remove the unbound conjugate, and 200 µl of substrate were added to each well including the blank, and the plate was incubated in the dark for 20 min. The enzymatic reaction was terminated with 50 µl of the stop solution and the plate read at 405 nm against a substrate blank. A standard curve was constructed with absorbance of histamine standard versus concentration in nanomolar (nM). The corresponding histamine concentration present in each sample was calculated from the standard curve.

Statistics

The two-tailed Student's t test for paired samples was used for determination of differences between the means of paired variables. One-way analysis of variance (ANOVA) without replications was used as a test for differences among the means of different groups of data. A p value of < 0.05 was considered significant.

	RESULTS

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The inhibitory effect of SP-A and SP-D on PHA- or Der p- allergen-stimulated lymphocyte proliferation was studied in asthmatic children (Figure 1). Surfactant proteins reduced [³H]thymidine incorporation in a dose-dependent manner. SP-A had a significant suppressive effect on PHA-stimulated lymphocyte proliferation at the concentration of 5 µg/ml as compared with PHA-stimulated cells alone (p < 0.05), while 10 µg/ml of SP-D was needed for a significant inhibitory effect on PHA-stimulated lymphocytes (Figure 1A). Similarly, Der p- stimulated lymphocyte proliferation was also suppressed in the presence of SP-A or SP-D, although as much as 10 µg/ml was needed to cause significant inhibition as compared with the Der p-stimulated lymphocytes alone (Figure 1B).

View larger version (18K): [in this window] [in a new window]   Figure 1.   The inhibitory effects of surfactant proteins on (A) PHA (1 µg/ml) and (B) Der p (10 µg/ml)- stimulated human PBMC [3H]thymidine incorporation in the presence of various concentrations of SP-A (solid bars) or SP-D (white bars). Each bar represents the mean ± SE of triplicate cultures. *p < 0.05 in the paired Student's t test in comparison with lymphocyte proliferation stimulated by mitogen plus surfactant protein and mitogen only.

This suppressive effect of surfactant proteins on PHA- or Der p-stimulated lymphocyte responses was observed in both asthmatic children in stable condition and age-matched control subjects, while SP-A, SP-D, and SP-D (N/CRD) had only a mild degree (< 25%) of inhibition on PHA- or allergen- induced lymphocyte responses in the samples taken from asthmatic children during an acute attack (Table 1).

Previously, we have reported that SP-D and its recombinant fragment could bind allergen in a calcium-dependent and lectin-specific manner (24). Therefore, the question arises of whether SP-A or SP-D's suppressive effects on PHA or Der p-induced lymphocyte proliferation may have resulted from the binding of surfactant protein to PHA or Der p. In order to address this question, the PBMC were pretreated with SP-A or SP-D for various time periods, washed three times with fresh culture medium, then their proliferation response to PHA or allergen was determined. Results in Figure 2A reveal that PBMC pretreated with SP-A, SP-D, and SP-D (N/CRD) for 12 h had lower responses of 50.6%, 56.2%, and 69.5% in PHA-stimulated [³H]thymidine incorporation, respectively. This inhibitory effect was also found in Der p-stimulated lymphocyte responses (Figure 2B). There was more than 50% inhibition of Der p-stimulated proliferation when PBMC were pretreated with SP-A or SP-D for 12 h as compared with no pretreatment. In this respect, the recombinant SP-D (N/CRD) was even more potent than native surfactant proteins. Thus, PBMC pretreated with this recombinant peptide for just 6 h showed 77.5% inhibition (Figure 2B).

View larger version (23K): [in this window] [in a new window]   Figure 2.   Pretreatment of surfactant proteins inhibits mitogen-induced PBMC proliferation. Incorporation of [3H]thymidine in cultures of human PBMC stimulated with (A) PHA (1 µg/ml) or (B) Der p (10 µg/ml) after pretreatment with SP-A (black bars), SP-D (white bars), and recombinent peptide of SP-D (hatched bars) at various incubation times (h). Each bar represents the mean ± SE of an experiment performed with replicates of six cultures (n = 3). *p < 0.05 in the paired Student's t test in comparison with lymphocyte proliferation stimulated by mitogen with and without surfactant protein pretreatment.

Surfactant proteins A and D are composed of basic units of CRD with collagen-like stalks, which specifically bind to mannose and maltose, respectively. To assess if the suppressive effect on lymphocyte proliferation is through the recognition of the glycosylation sites on lymphocyte surface molecules, competition assays were performed in the presence or absence of 100 mM mannose (for SP-A) or maltose (for SP-D). Glucose was used as a nonspecific sugar inhibition control. Table 2 shows that, in addition to the intact, native, surfactant protein A and D molecules, the recombinant fragment, which is composed of neck and CRD of SP-D [SP-D (N/CRD)], also has the same suppressive effect on lymphocyte proliferation. The inhibitory effect was abolished in the presence of 100 mM mannose or maltose, but not in the presence of glucose. These results suggest that the CRD region of SP-A and SP-D may interact with the carbohydrate structures on the surface molecules of the PBMC.

The interaction of surfactant proteins with the cell surface molecules, and the possibility that surfactant proteins can activate monocytes during cell proliferation were investigated by flow-cytometry analysis (Figure 3). We found that PBMC pretreated with SP-D (N/CRD) recombinant peptide for 12 h had a dose-dependent decrease in staining of FITC-labeled anti-CD11b on their surface as compared with basal staining content (54.9% versus 90.1%), while the staining of FITC-labeled anti-HLA-DR of gated monocytes did not significantly change when incubated with 10 µg/ml of recombinant peptide (83.4% versus 95.4%).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Flow-cytometry analysis of the staining of FITC-labeled anti-human CD11b (left column) and HLA-DR (right column) antibodies on monocytes, when cells were incubated with SP-D (N/CRD) recombinant peptide at the concentrations of 0.1 µg/ml (first row), 1 µg/ml (second row), and 10 µg/ml (third row). Shadow tracing in the background represents basal staining content without SP-D (N/CRD). There was a dose-dependent decrease in staining of FITC-labeled anti-CD11b incubated with SP-D (N/CRD) (bold tracing) as compared with basal staining content (54.9% versus 90.1%), while anti-HLA-DR staining was reduced only by 12% when PBMC were incubated with 10 µg/ml of recombinant peptide. Results of flow-cytometry analysis have been repeated three times, and the standard deviation of each concentration tested was within 5% of the mean value. MFI = mean fluorescence intensity.

In order to explore the role of surfactant proteins in the immediate-type hyersensitivity reaction of histamine release, diluted whole blood of asthmatic children and control subjects was challenged with Der p allergen in the presence of surfactant proteins. As seen in Figure 4, SP-A, SP-D, and SP-D (N/CRD) were able to inhibit Der p-stimulated histamine release in a dose-dependent manner. Surfactant proteins, at the concentration of 10 µg/ml, could reduce histamine release induced by Der p stimulation by more than 50% (Figure 5). However, pretreatment of PBMC with a maximal concentration (50 µg/ml) of surfactant proteins did not have any inhibitory effect on histamine release prompted by Der p stimulation (data not shown). These results suggest that lung surfactant proteins A and D inhibit histamine release from the sensitized basophils of asthmatic children probably through their binding with glycosylated allergens and making them unavailable for cross-linking the specific IgE molecules on the histamine-releasing cells.

View larger version (17K): [in this window] [in a new window]   Figure 4.   Inhibitory effects of surfactant proteins on Der p-stimulated histamine release. Histamine release from the Der p (10 µg/ ml)-stimulated whole blood of mite-sensitive asthmatic children in the presence of various concentrations of SP-A (circles), SP-D (squares), and SP-D (N/CRD) (triangles). Each point represent the mean ± SE of a set of triplicate culture assays (n = 3). *p < 0.05 in the paired Student's t test in comparison with histamine release stimulated by Der p plus surfactant protein and Der p only.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Inhibitory effects of SP-A (10 µg/ml) and SP-D (10 µg/ ml) on various concentrations of Der p-dependent histamine release. Each point represents the mean ± SE of an experimental condition in triplicate culture assays (n = 3). Both SP-A and SP-D, at the concentration of 10 µg/ml, could reduce Der p (from 1.25 to 10 µg/ml)-dependent histamine release by more than 50%.

	DISCUSSION

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We have demonstrated that purified surfactant proteins and a recombinant fragment, composed of neck and CRD of SP-D, were able to inhibit histamine release of early type hypersensitivity reaction, in a dose-dependent manner, in the whole blood of children with asthma, whereas pretreatment of PBMC with surfactant proteins did not have a suppressive effect on Der p- induced histamine release. Previously, we have reported that purified native SP-D and SP-D (N/CRD) were able to bind Der p through their CRDs in a calcium-dependent manner and prevent its interaction with specific IgE in the Der p sensitive asthmatic serum (24). Therefore, we conclude that this inhibitory effect of surfactant proteins on histamine release may have resulted from their binding with allergen, thus preventing its cross-linking with IgE molecules on histamine-releasing cells. Our result also is consistent with the finding that lung surfactant proteins A and D are able to inhibit histamine release of basophils obtained from patients with allergic bronchopulmonary aspergillosis (27).

Moreover, we also found that surfactant proteins had a suppressive effect on allergen-stimulated lymphocyte proliferation. This proliferation of stimulated lymphocytes is important for the development of late-phase allergic inflammation triggered by Th2-type cytokine release and eosinophil recruitment (28). In the study reported here, we effectively ruled out the possibility that the inhibition was through the interaction of surfactant protein with mitogen or allergen. In our hands, PBMC pretreated with surfactant protein for various time periods showed an inhibited proliferation response to PHA or Der p stimulation in a time- and dose-dependent manner (Figure 2).

The mechanism of suppression of lymphocyte proliferation is not yet clear. Surfactant has been shown to modulate the functions of pulmonary inflammatory cells (18). It has been recognized that pulmonary lymphocytes are hyporesponsive to in vitro stimulation with IL-2 in the presence of pulmonary surfactant, and that surfactant is able to inhibit IL-2-induced proliferation and the generation of lymphokine-activated killer cells (29). While SP-A and SP-D enhance various functions of alveolar macrophages, such as binding, opsonization, and phagocytosis of infectious agents (14), surfactant phospholipids seem to be able to downregulate local lymphocyte activities (30). Kremlev and Phelps (4, 5) found that surfactant phospholipids of dipalmitoylphosphatidylcholine (DPPC) and Surventa inhibited lymphocyte proliferation when concanavalin A was used as a mitogen. However, SP-A enhanced concanavalin-A-dependent proliferation and stimulated the production of inflammatory cytokines by human PBMC. In contrast, Borron and coworkers (31) reported that SP-A was able to suppress both PHA- and anti-CD3-activated proliferation of PBMC of normal individuals, and SP-A could inhibit the production of IL-2 by human lymphocytes stimulated by PHA. They suggested that SP-A may bind to integrin adhesion molecules on the surface of lymphocytes, such as leukocyte function-associated antigen 1 (LFA-1), which may mediate costimulation of T-cell proliferation (31). These integrin adhesion molecules contain mannose-linked oligosaccharides to which SP-A is known to have specific affinity (15). The reasons for these conflicting results of SP-A on lymphocyte functions remain to be determined. They may be attributable to the variable nature of the surfactant preparation, differences in purification methods of SP-A (32), and the cell types and mitogens studied.

Our results tend to support the interpretation suggested by Borron and coworkers (31), because in addition to the native surfactant proteins, the recombinant fragment of neck and CRD regions of SP-D also have the suppressive effect on lymphocyte proliferation. Furthermore, this suppressive effect was abolished in the presence of specific sugars in the culture medium (Table 1). One of the possible mechanisms of suppressive effect of surfactant proteins on lymphocyte proliferation may be that by binding to monocytes/macrophages, surfactant protein may activate the production of their suppressive factors to inhibit lymphocyte proliferation. We have used flow-cytometry analysis to differentiate these possibilities. The expression of an activated surface marker of monocytes, HLA-DR, did not increase significantly after incubation with surfactant proteins for various time periods (Figure 3). On the contrary, the staining of FITC-labeled anti-CD11b (-chain of CR3 integrin) in PBMC had a dose-dependent decrease in manner when incubated with SP-D (N/CRD) recombinant peptides (Figure 3). These results strongly suggest that surfactant proteins, through their CRDs, can suppress lymphocyte proliferation by direct interaction with integrin on the surface of lymphocytes. At the present moment, there is no direct evidence that surfactant proteins can suppress lymphocyte proliferation through activating monocytes.

Recently, van der Graaf and coworkers (10) reported decreased levels of SP-A in the bronchoalveolar lavage fluid of asthmatics in comparison with healthy control subjects. Reduced SP-A levels in asthmatics could reflect an impaired resistance against pulmonary inflammation, with further disturbance of the surfactant system. It may be hypothesized that the immunomodulatory effects of surfactant contribute to the downregulation of airway inflammation during acute asthmatic reactions. It is interesting to observe that during an acute asthmatic attack, the downregulatory effect of surfactant proteins on T-cell proliferation was minimized to 24%, significantly lower than those in the stable asthmatic children and control subjects (Table 2). This phenomenon may be due to the overexpression of integrin adhesion molecules on the surface of activated T cells during an asthmatic attack (33), which decreases the suppressive effect of surfactant proteins.

Cytokines such as interleukin-1 (IL-1) and TNF- are involved in airway inflammation and are indeed elevated in asthma (34). Both IL-1 and TNF- are potent inducers of the transcription factor, nuclear factor-B (NF-B). It was recently found that surfactant suppresses NF-B activation in human monocytic cells (35). Thus, the inhibitory effect of surfactant on inflammatory cytokine production may involve transcript regulation through the inhibition of NF-B activation. However, delineation of the precise role of surfactant in the immunoregulatory network of asthmatic airway inflammation awaits further study.

Taken together, SP-A and SP-D are able to suppress allergen-induced lymphocyte proliferation and histamine release in asthmatic children who are in stable condition. These effects may be mediated by two different mechanisms. On the one hand, SP-A and SP-D may bind to allergen and hinder its interaction with surface-bound IgE molecules and reduce the level of histamine release from sensitized basophils; on the other hand, surfactant proteins may bind to surface molecules on the cell membrane of lymphocytes and thus induce inhibitory signals that affect the transduction of lymphocyte proliferation. When T cells have already been activated during an asthmatic attack, this inhibitory effect of surfactant is nulled because the surface molecules of activated lymphocytes are overexpressed. Moreover, there are generally decreased amounts and functions of surfactant proteins in allergic inflammation. Because histamine release and lymphocyte proliferation are two essential steps in the development of asthmatic symptoms, our findings that SP-A and SP-D are able to inhibit both reactions implicate the important protective role of these two substances in the pathogenesis of asthma.