Surfactant protein A is defective in abrogating inflammation in asthma

Abstract

Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the ability of SP-A derived from normal and asthmatic subjects to modulate the inflammatory response elicited by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. Fourteen asthmatic and 10 normal control subjects underwent bronchoscopy with airway brushing and bronchoalveolar lavage (BAL). Total SP-A was extracted from BAL. The ratio of SP-A1 to total SP-A (SP-A1/SP-A) and the binding of total SP-A to M. pneumoniae membranes were determined. Airway epithelial cells from subjects were exposed to either normal or asthmatic SP-A before exposure to M. pneumoniae. IL-8 protein and MUC5AC mRNA were measured. Total BAL SP-A concentration did not differ between groups, but the percentage SP-A1 was significantly increased in BAL of asthmatic compared with normal subjects. SP-A1/SP-A significantly correlated with maximum binding of total SP-A to M. pneumoniae, but only in asthma. SP-A derived from asthmatic subjects did not significantly attenuate IL-8 and MUC5AC in the setting of M. pneumoniae infection compared with SP-A derived from normal subjects. We conclude that SP-A derived from asthmatic subjects does not abrogate inflammation effectively, and this dysfunction may be modulated by SP-A1/SP-A.

Fig. 1.

Ratio of surfactant protein A (SP-A)1 to total SP-A (SP-A1/SP-A) is increased in the bronchoalveolar lavage (BAL) from asthmatic subjects. In vitro-expressed SP-A1 variant 6A² and total SP-A, purified from a normal subject BAL of known concentration, were used as standards. SP-A1 and total SP-A were measured via ELISA using SP-A1 and total SP-A specific antibodies. Data are expressed as means ± SE. *P < 0.01 between normal and asthma subjects.

Fig. 2.

SP-A binding to solid-phase mycoplasma membranes is markedly reduced in asthmatic subjects. One hundred nanograms of mycoplasma membranes were adsorbed to microtiter wells in 0.1 mM NaHCO₃, pH 9.6. The concentration-dependent binding of normal (NSP-A) and asthmatic SP-A (ASP-A) to solid-phase Mycoplasma pneumoniae membranes (SP-A bound) was detected by ELISA. Area under the curve (AUC) analysis of the SP-A binding curves revealed a significantly lower AUC for ASP-A curves compared with NSP-A, P = 0.03.

Fig. 3.

SP-A variants exhibit different binding properties to mycoplasma membranes. One hundred nanograms of mycoplasma membranes were adsorbed to microtiter wells in 0.1 mM NaHCO₃, pH 9.6. Purified SP-A (100 ng) from the subjects with alveolar proteinosis (APP), or recombinant proteins expressed in HEK-293 cells, [50V, 219R] SP-A1 and [91A, 223Q] SP-A2 (Hu SPA-1 and Hu SPA-2, respectively), were incubated with the solid-phase membranes for 2 h at 37°C. The bound proteins were detected by ELISA with horseradish peroxidase-conjugated polyclonal anti-SP-A antibody. Orthophenylenediamine (1 mg/ml) was used as the color development reagent. Nonspecific binding was blocked at each step of the procedure by using a buffered 3% bovine serum albumin. Values shown are means ± SE. *P < 0.05.

Fig. 4.

SP-A from asthmatic subjects is defective in attenuating MUC5AC mRNA expression by asthmatic epithelial cells following M. pneumoniae infection. MUC5AC mRNA expression from airway epithelial cells isolated from normal control and asthmatic subjects exposed to M. pneumoniae at 50 cfu/cell (MP50) alone and after exposure to NSP-A (MP50 + NSP-A), ASP-A (MP50 + ASP-A), each at 20 μg/ml, and a combination of both NSP-A and ASP-A (MP50 + NSP-A+ASP-A). The data, representing fold change from negative, uninfected control, are expressed as means ± SE. *P < 0.01 between MP50 in normal vs. asthmatic subjects, †P < 0.01 between MP50 and MP50 + NSP-A; ‡P < 0.01 between MP50 and MP50 + ASP-A; #P < 0.01 between MP50 and MP50 + NSP-A+ASP-A; #P < 0.05 between MP50 + NSP-A and MP50 + ASP-A.

Fig. 5.

SP-A from asthmatic subjects is defective in abrogating IL-8 levels secreted by asthmatic epithelial cells following M. pneumoniae infection. IL-8 expression from airway epithelial cells from normal control subjects and subjects with asthma exposed to M. pneumoniae at 50 cfu/cell (MP50) alone, and after exposure to NSP-A (MP50 + NSP-A), ASP-A (MP50 + ASP-A), each at 20 μg/ml, and a combination of both NSP-A and ASP-A (MP50 + NSP-A+ASP-A). The data, representing the ratio to negative, uninfected control are expressed as means ± SE. *P < 0.01 between MP50 and MP50 + NSP-A; †P < 0.01 between MP50 and MP50 + NSP-A + ASP-A; #P < 0.05 between MP50 + ASP-A and MP50 + NSP-A and between MP50 + ASP-A and MP50 + NSP-A+ASP-A.

Fig. 6.

Reduction of MUC5AC and IL-8 by NSP-A in the setting of mycoplasma infection is specific to SP-A. Purified NSP-A mixed with mannose-Sepharose beads in the presence of 2 mM CaCl₂ (NSP-A + Ca) and SP-A mixed with mannose-Sepharose beads in the presence of 2 mM EDTA (NSP-A + EDTA) was added separately to airway epithelial cells from 4 asthmatic subjects alone or in the presence of M. pneumoniae at 50 cfu/cells (MP50). A: fold change in MUC5AC mRNA relative to negative, uninfected control. B: ratio of IL-8 protein to negative, uninfected control. The data, representing the ratio to negative, uninfected control, are expressed as means ± SE. *P < 0.01 compared with MP50.

Fig. 7.

Oligomerization status of purified SP-A. Three NSP-A samples and 3 ASP-A samples were purified and subjected to 4–20% PAGE under nonreducing conditions (A) or under native conditions (B) followed by silver staining. Numbers on left indicate the molecular mass. Marks on right represent oligomers.

Fig. 8.

Oxidation levels of purified SP-A from normal and asthmatic subjects. Two hundred nanograms of purified SP-A from normal and asthmatic subjects, with or without DNase and RNase pretreatment, were blotted onto nitrocellulose membrane and oxidized proteins were detected by the OxyBlot method. Blots were exposed to XAR films following enhanced chemiluminescent detection and quantified by densitometry. The data representing the percentage of optical density of the NSP-A without treatment with DNase and RNase are expressed as means ± SE.