Phospholipid molecular species of bronchoalveolar lavage fluid after local allergen challenge in asthma

Emma L. Heeley1, Jens M. Hohlfeld2, Norbert Krug2, and Anthony D. Postle1

1 Child Health, University of Southampton, Southampton SO16 6YD, United Kingdom; and 2 Department of Respiratory Medicine, Hannover Medical School, D-30625 Hannover, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrospray ionization mass spectrometry was used to quantify phosphatidylcholine (PC) and phosphatidylglycerol (PG) molecular species in bronchoalveolar lavage fluid (BALF) from control and mild asthmatic subjects after local allergen challenge. BALF was obtained from 5 control and 13 asthmatic subjects before and 24 h after segmental allergen and saline challenge. There were no differences in the ratio of total PC to total PG or in the molecular species composition of PC or PG between the asthmatic and control groups under basal conditions. Allergen challenge in asthmatic but not in control volunteers caused a significant increase in the PC-to-PG ratio because of increased concentrations of PC species containing linoleic acid (16:0/18:2 PC, 18:0/18:2 PC, and 18:1/18:2 PC). These molecular species were characteristic of plasma PC analyzed from the same subjects, strongly suggesting that the altered PC composition in BALF in asthmatic subjects after allergen challenge was due to infiltration of plasma lipoprotein, not to catabolism of surfactant phospholipid. Interactions between surfactant and lipoprotein infiltrate may contribute to surfactant dysfunction and potentiate disease severity in asthma.

serum lipoprotein; lung surfactant; phosphatidylcholine; phosphatidylglycerol; electrospray ionization mass spectrometry


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EXTRAVASATION OF PLASMA COMPONENTS into the airways has been strongly implicated in the pathology of both early and late responses to allergens in asthma (for a review, see Ref. 18). This exudation of protein-rich fluid onto the airway mucosal surface has been proposed to contribute to the increased airway resistance in asthma by one of two mechanisms. Fluid collecting in epithelial ridges may reduce airway cross-sectional area and so amplify the reduction in airway gas flow (22), and inhibitory components in the exudate may impair the surface properties of pulmonary surfactant (9, 21). Surfactant is distributed at the air-liquid interface throughout the lungs as demonstrated both by electron-microscopic studies (10) and by the isolation of functional surfactant from pig trachea (4), and the surface pressure generated by surfactant has been proposed as a critical force to maintain the patency of the smaller bronchioles and terminal airways. In this model, inactivation of surfactant function by plasma protein would contribute to the edema, mucus plugging, and airway collapse characteristic of the acute asthmatic response (8).

This concept is supported by studies that have induced anaphylactic bronchoconstriction in immunized guinea pigs in response to inhalation of ovalbumin antigen. The influx of fluid and protein into the airway lumen was associated with increased minimal surface tension (gamma min) of bronchoalveolar lavage fluid (BALF) as measured with a pulsating bubble surfactometer (15), and both bronchoconstriction and edema could be prevented by pretreatment of animals with exogenous surfactant (3, 20). In human studies, gamma min measured in induced sputum samples increased in acute asthma and subsequently returned to normal low values during the recovery phase (13). There has been one report of the successful treatment of acute asthma by exogenous surfactant (14), but so far there have been no reported controlled clinical trials to test the efficacy of surfactant therapy for asthma.

The mechanisms responsible for surfactant inhibition in the asthmatic response have not been clearly defined. Infiltration of plasma proteins such as fibrin monomer (21) into the airways has generally been proposed to cause a reversible inhibition of surface properties of surfactant. This concept is supported by observations that removal of such soluble inhibitors by washing the surfactant fraction in vitro could substantially restore surfactant function (12). It is possible that other components of lung edema fluid might also contribute to surfactant dysfunction in asthma, but no such additional factors have yet been evaluated.

Hohlfeld et al. (12) have previously reported that both total protein and gamma min in BALF increased 24 h after local allergen challenge and that these responses were not apparent in either sham-challenged asthmatic or allergen-challenged control subjects. In this study, we have employed flow injection electrospray ionization mass spectrometry (ESI-MS) (19) to determine whether these changes to surfactant function were accompanied by any modification in the phospholipid composition in BALF after local allergen challenge in mild asthmatic subjects.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Study protocol. BALF samples were obtained by fiber-optic bronchoscopy from control and asthmatic subjects before and after local administration of sham or allergen challenge (for details, see Ref. 12). At the first bronchoscopy, we collected BALF for baseline values from one lobe and challenged another lobe with saline solution only and a lobe in the other lung with antigen. BALF was then obtained from both challenged lobes by a second bronchoscopy 24 h later. Nine healthy volunteers and fifteen patients with mild asthma were enrolled. All patients had allergic asthma according to the criteria from the National Heart, Lung, and Blood Institute (National Institutes of Health, Bethesda, MD) (17). On the day of bronchoscopy, a skin wheal dose series was obtained with either Dermatophagoides pteronyssinus or grass pollen depending on which produced the largest response. The concentration used for endobronchial challenge was one-tenth of that which elicited a skin wheal with a diameter of 3 mm. Current medication consisted of beta 2-agonists only, and no patient had used glucocorticosteroids, sodium chromoglycate, or theophylline for at least the previous 6 wk.

The healthy volunteers had no history of allergic diseases or any other disorders; they had negative skin prick tests, normal IgE levels (<100 IU/ml), normal lung function tests, and no bronchial hyperresponsiveness. All study subjects were nonsmokers, and none had suffered an episode of acute bronchitis during the 4-wk period preceding the challenge. The study was approved by the Ethic Committee of Hannover Medical School (Hannover, Germany), and informed consent was obtained from each person in the study.

Segmental allergen challenge and collection of BALF. The procedures for fiber-optic bronchoscopy and segmental allergen challenge used in this study have been previously described in detail (12). The bronchoscope (P30, Olympus Optical, Tokyo, Japan) was wedged into the inferior lingular bronchus, which was then lavaged with five aliquots of 20 ml of warmed saline to obtain the initial BALF sample. The bronchoscope was then passed into the superior lingular bronchus, and 10 ml of warmed saline were instilled as control challenge. Finally, the instrument was moved into the medial segment of the middle lobe of the contralateral lung where 10 ml of warm allergen solution were instilled. Twenty-four hours after this initial bronchoscopy, the subjects underwent a second bronchoscopy to obtain BALF from the sham and allergen-challenged lobes. BALF aliquots from each instillation were pooled, filtered through sterile gauze, and then centrifuged at 250 g for 10 min. An aliquot of the cell-free supernatant was stored at -28°C until further analysis. The remainder of the BALF supernatant was centrifuged at 48,000 g for 60 min to pellet large-aggregate (LA) fractions. Venous blood samples (10 ml) were taken on both days and allowed to clot, and the serum was stored at -28°C until analysis.

Analysis of phospholipid and protein. The concentration of total phospholipid in BALF supernatant was determined as phospholipid phosphorus with the method of Bartlett (2) after extraction of a lipid fraction with chloroform and methanol (5). Protein concentration was determined according to Lowry et al. (16). All assays were performed in duplicate, and the mean value is reported.

Preparation of samples for MS. Aliquots of BALF containing 25 nmol of phospholipid phosphorus were extracted with chloroform and methanol according to Bligh and Dyer (5). Internal standards of dimyristoylphosphatidylglycerol (14:0/14:0 PG, 1 nmol) and dimyristoylphosphatidylcholine (14:0/14:0 PC, 5 nmol) were added before extraction. Aliquots of serum (50 µl) were extracted with the same method after the addition of 15 nmol of 14:0/14:0 PC as an internal standard. All lipid extracts were dried under a stream of nitrogen gas and stored at -20°C until analysis. All phospholipid standards were obtained from Sigma-Aldrich (Poole, UK).

MS. All MS was performed with a triple quadrapole instrument (Fisons VG Quattro II, Micromass UK, Manchester, UK) fitted with either an electrospray or a nanoflow interface. Molecular species of PC were preferentially detected with positive-ionization conditions, whereas PG species were detected under negative-ionization conditions (19). Molecular species are designated as n:a/m:b, where n and m are the number of carbon chains at the sn-1 and sn-2 positions, respectively, and a and b are the number of double bonds in the chains. Species were identified by the mass-to-charge ratio (m/z), which for these singly charged phospholipid molecules was equivalent to molecular mass.

ESI-MS. Dried lipid extracts of BALF were dissolved in 25 µl of the injection solvent methanol-chloroform-water (7:2:1 vol/vol) containing 1% (wt/vol) NH4OH. NH4OH was added to enhance the negative-ion response and to increase the formation of molecular ions in the positive-ion mode by displacing sodium ions that tend to associate with PC species (sodiated species). A sample aliquot (5 µl) was introduced into the capillary of the electrospray interface by a rheodyne valve injection into the methanol-chloroform-water (7:2:1 vol/vol) containing 1% NH4OH (wt/vol) pumped at a flow rate of 50 µl/min. Spectra were acquired in both positive and negative ionization on alternating scans between m/z 400 and 900, with a scan time of 2 s and a resolution of 0.1 mass unit. Capillary voltages of +3 and -2.5 kV, with cone voltages of +48 and -77 V, respectively, were applied.

PC composition of the serum samples was analyzed by an analogous technique except that sodium acetate was used in the injection solvent to increase the formation of the sodiated PC species. This modification was possible because no measurement of acidic phospholipids in a negative-ionization mode was made for these samples. Serum samples were dissolved in 75 µl of methanol-chloroform-water (7:2:1 vol/vol) containing 20 mM sodium acetate, and an aliquot (2.5 µl) was introduced into the ESI-MS by injection into the methanol-chloroform-water (7:2:1 vol/vol) pumped at a flow rate of 50 µl/min. Spectra were collected only in the positive-ionization mode with the conditions described above.

All spectral data were collected in continuum format, averaged over a scan period of 1 min, and then analyzed with MassLynx software (Micromass UK). These continuum data for each ion peak were transformed into a centroid format on the basis of peak area. The resulting centroid peak height values for individual phospholipid molecular species were corrected for the 13C component. The fractional (percentage) composition and the absolute (nmol/ml) concentration of the phospholipid species were then calculated by reference to the response of the relevant internal standard.

Tandem MS-MS. Tandem MS-MS was performed to confirm the identity of the various mass ions. Lipid extracts of the samples were prepared as in Preparation of samples for MS and then dissolved in 25 µl of methanol-chloroform (1:2 vol/vol) containing 1% NH4OH. An aliquot (10 µl) of the sample solution was loaded into a borosilicate tip and then attached to a nanoflow probe (Micromass UK). The sample was then gently ionized with capillary voltages of approximately +1.2 and -1.35 kV and cone voltages of +30 and -40 V in the positive and negative modes, respectively. Fragmentation was induced with argon gas, typically at 2.7 × 10-3 mbar with a collision energy of 30 eV.

Surface tension analysis. Surface activity of BALF was measured with a pulsating bubble surfactometer as previously described (12). The LA fraction of BALF was resuspended to 1 mg/ml, and 40 µl of this suspension were used for surface tension analysis. The gamma min is the value at minimal bubble size registered after 5 min of pulsation at 20 cycles/min at a temperature of 37°C.

Statistical analysis. The results expressed in Tables 1 and 2 are median values with the ranges indicated. In Figs. 1-7, they are median values with interquartile and 95% confidence intervals indicated. Statistical differences between initial, sham, and allergen-exposed groups were compared with a Friedman test for both control and asthmatic groups. Wilcoxon tests were then performed on paired data when P values were <0.05. Selected parameters of BALF phospholipid composition were compared with protein concentration and surface tension measurements with Pearson's correlation.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Typical electrospray ionization mass spectra of the phospholipid components in BALF are shown for PC under positive ionization (Fig. 1A) and for PG under negative ionization (Fig. 1B). PC species were identified as the M+, and absolute amounts were calculated relative to the internal standard 14:0/14:0 PC (m/z 678). PG species were identified as the [M-H]- and quantified relative to the internal standard 14:0/14:0 PG (m/z 665). Identities of molecular species were confirmed by tandem MS-MS of the various mass ions. With the use of negative ionization, PC species as chloride adducts and PG species both fragmented to generate ions corresponding to the fatty acid anions and dehydrated glycerophosphate (m/z 153). Using authentic standards, it has been shown (11) that such fragmentation can provide unambiguous structural information because the intensity of fatty acid anions produced from the sn-2 position was double that from the sn-1 position. For instance, Fig. 2A shows the 16:0 (m/z 255) and 18:1 (m/z 281) fatty acid anion ions produced by fragmentation of m/z 747, confirming this structure as 16:0/18:1 PG. Tandem MS-MS was particularly useful for the analysis of different molecular species with the same molecular mass. For instance, the predominant fragmentation product of m/z 773 was the fatty acid anion 18:1 (m/z 281), with smaller amounts of 18:2 (m/z 279) and 18:0 (m/z 283; Fig. 2B). This confirmed that the major molecular species component of the m/z 773 ion was 18:1/18:1 PG, with ~30% 18:0/18:2 PG.


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Fig. 1.   Analysis of bronchoalveolar lavage fluid (BALF) phosphatidylcholine (PC) and phosphatidylglycerol (PG) molecular species by electrospray ionization mass spectrometry. Molecular species are designated as n:a/m:b, where n and m are no. of carbon chains at sn-1 and sn-2 positions, respectively, and a and b are no. of double bonds. A: under positive-ionization mode, only PC molecular species were seen and analyzed relative to internal standard 14:0/14:0 PC (m/z 678, which is equivalent to molecular mass). PC molecular species analyzed were 14:0/16:0 PC (m/z 706), 16:0/16:1 PC (m/z 732), 16:0/16:0 PC (m/z 734), 16:0/18:2 PC (m/z 758), 16:0/18:1 PC (m/z 760), 18:1/18:2 PC (m/z 784), and 18:1/18:1 PC plus 18:0/18:2 PC (m/z 786). B: PG species were analyzed under negative-ionization mode relative to internal standard 14:0/14:0 PG (m/z 665). PG species analyzed were 16:0/16:0 PG (m/z 721), 16:0/18:2 PG (m/z 745), 16:0/18:1 PG (m/z 747), 18:1/18:2 PG (m/z 771), 18:1/18:1 PG plus 18:0/18:2 PG (m/z 773), and 18:1/18:2 PG (m/z 775).



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Fig. 2.   Tandem mass spectrometry (MS)-MS was used to identify molecular species configurations. A: identity of m/z 747 ion was confirmed as 16:0/18:1 PG. Fragmentation of m/z 747 generated an 18:1 fatty acid anion at m/z 281 with twice the intensity of that of 16:0 fatty acid anion at m/z 255. B: similarly, isobaric components at m/z 773 were identified as 18:1/18:1 PG, with ~30% 18:0/18:2 PG because predominant product (inset) was 18:1 fatty acid anion (m/z 281), with smaller amounts of 18:2 (m/z 279) and 18:0 (m/z 283) anions.

Total concentrations of PC and PG were calculated by the addition of all individual molecular species measured. The ratio of total PC to PG in BALF (Fig. 3) did not change in the control group after either sham or allergen challenge or in the asthmatic group after sham challenge. However, the PC-to-PG ratio increased significantly 24 h after allergen challenge in asthmatic subjects (P < 0.05), and comparison of absolute concentrations suggested that the increase was due to an increase in total PC. Total PC after allergen challenge (25.1 nmol/ml) was significantly greater (P < 0.05) than after sham challenge (19.1 nmol/ml), with no difference between the values for PG (5.3 and 5.5 nmol/ml, respectively).


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Fig. 3.   Ratio of total PC to PG in BALF from control and asthmatic subjects before (initial) and after sham and allergen challenges. Total concentrations of PC and PG were calculated by addition of all individual molecular species. Thick horizontal lines, median values; boxes, interquartile ranges; vertical lines, 95% confidence limits; +, extreme values. * P < 0.05 for allergen vs. initial samples from asthmatic subjects.

The molecular species composition of PC in BALF under baseline conditions was identical for both the control (Fig. 4A) and asthmatic (Fig. 4B) groups, and this was not altered in the control subjects after either sham or allergen challenge or in the asthmatic subjects after sham challenge. In contrast, there were significant changes to the PC composition in BALF from asthmatic subjects after allergen challenge (Fig. 4B); the fractional concentration of 16:0/16:0 PC decreased significantly and also displayed a much larger range of individual values (median 36.6%, range 13.8-50.7%, after challenge compared with median 47.1%, range 40.1-54.7%, before allergen challenge). This decreased 16:0/16:0 PC in asthmatic subjects after allergen challenge was accompanied by corresponding increased concentrations in BALF of PC species containing 18:2 (Fig. 4B). The identities of these species were confirmed by tandem MS-MS as principally 16:0/18:2 PC, 18:1/18:2 PC, and 18:0/18:2 PC.


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Fig. 4.   Composition of individual PC molecular species in BALF for control (A) and asthmatic (B) subjects before and after sham and allergen challenges. Thick horizontal lines, median values; boxes, interquartile ranges; vertical lines, 95% confidence limits; +, extreme values. * P < 0.05 for allergen vs. initial samples from asthmatic subjects.

The compositions of individual molecular species of PG in BALF are summarized in Table 1. Although this composition varied considerably between individuals, the same major molecular species were predominant, namely 16:0/18:1 PG and 18:1/18:1 PG, with smaller proportions of 18:0/18:2 PG and 18:0/18:1 PG. Median values for the PG species composition were identical for control and asthmatic subjects and remained unchanged in both groups after either challenge procedure.

                              
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Table 1.   BALF PG molecular species composition

Because plasma phospholipid is characterized by PC species containing 18:2, we investigated whether the increased content of 18:2-containing PC species in BALF from asthmatic subjects after allergen challenge could have been derived from infiltration of plasma lipoproteins into the airways. Table 2 documents the PC molecular species composition in serum from both control and asthma groups. The major PC species present in serum were 16:0/18:2 PC, 16:0/18:1 PC, 16:0/20:4 PC, 18:1/18:2 PC, and18:0/18:2 PC. In contrast to BALF PC, there were negligible amounts of 16:0/16:0 PC in any serum sample. Serum PC composition for both groups was unchanged the day after segmental challenge.

                              
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Table 2.   Serum PC molecular species composition

We then compared the PC mass spectrum of BALF from an asthmatic subject challenged with allergen that demonstrated the most extreme changes (Fig. 5A) with that of serum taken on the same day (Fig. 5B). PC species characteristic of serum were enriched in this BALF sample after challenge, whereas species typical of surfactant were decreased. For instance, the presence of large amounts of 18:2-containing species in both traces, namely 16:0/18:2 PC and 18:0/18:2 PC, was consistent with the infiltration of plasma-derived PC into the BALF. Compared with the BALF before allergen challenge (Fig. 1A), 16:0/16:0 PC was no longer the major species after challenge.


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Fig. 5.   Mass spectra of PC from BALF (A) and plasma (B) from the same asthmatic subject after allergen challenge. This subject exhibited an extreme response, and results clearly show presence in BALF of 18:2-containing PC species, typical of plasma PC.

To evaluate whether this decreased 16:0/16:0 PC in BALF from asthmatic subjects after allergen challenge could be due to plasma infiltration, the concentration of 16:0/16:0 PC as an index of lung surfactant was compared with that of 16:0/18:2 PC as an index for plasma phospholipid (Fig. 6). There was a strong inverse correlation between the relative decrease in 16:0/16:0 PC and the increase in 16:0/18:2 PC above the baseline value of ~10%, which was the normal content of 16:0/18:2 PC in lung surfactant. This result was consistent with the hypothesis that the decreased 16:0/16:0 PC in BALF from asthmatic subjects 24 h after allergen challenge was probably due to dilution of surfactant PC with plasma-derived lipoprotein PC from edema fluid rather than to altered composition of surfactant phospholipid.


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Fig. 6.   Assessment of extent of plasma phospholipid infiltration in BALF from asthmatic subjects after allergen challenge. Percent contents of 16:0/16:0 PC and 16:0/18:2 PC were correlated as markers for surfactant and plasma phospholipid, respectively. Dashed lines, median values from control subjects after allergen challenge.

Hohlfeld et al. (12) have previously reported that allergen challenge to these asthmatic patients increased the protein concentration in BALF 24 h later. This was accompanied by impaired surface tension, indicated by an increase in gamma min of the LA fraction as measured with a pulsating bubble surfactometer. Consequently, we correlated the 16:0/18:2 PC content in BALF, as a marker for plasma infiltration, with both these parameters in asthmatic subjects after allergen challenge. There were strong correlations of percent 16:0/18:2 PC with both protein concentration (P < 0.001; Fig. 7A) and gamma min (P < 0.001; Fig 7B). The comparison with protein concentration in BALF supports our suggestion that increased 16:0/18:2 PC is potentially a sensitive marker for plasma infiltration of the airways in asthma.


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Fig. 7.   Fractional concentration of 16:0/18:2 PC in BALF from asthmatic subjects after allergen challenge was correlated with total protein concentration (A) and minimal surface tension (gamma min; B) of large-aggregate fraction.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we measured for the first time the molecular species composition of PC and PG in BALF after local allergen challenge in asthmatic and control subjects. These PC and PG compositions before challenge were identical for both subject groups and were characteristic of purified surfactant. After allergen challenge, the increased concentrations of unsaturated PC species in the asthmatic group but not in the control group (Fig. 4B) was striking. Comparison of molecular species composition suggested strongly that this altered PC composition was due to infiltration of plasma phospholipid components (Table 2, Fig. 5). The compositions of both serum PC and the increased BALF PC after allergen challenge were both dominated by 18:2-containing species, principally 16:0/18:2 PC. This increased content of 18:2-containing species evidently contributed to the decrease in the percent concentration of 16:0/16:0 PC in asthmatic subjects after local allergen challenge. It is also possible, however, that surfactant PC was altered under these conditions, especially because the decrease in 16:0/16:0 PC was greater than the increase in 16:0/18:2 PC. This suggestion agrees with the results from another study in our laboratory (S.M. Wright and P.M. Hockey, unpublished observations) that found a progressive decrease in the fractional concentration of 16:0/16:0 PC in induced sputum samples from asthmatic subjects with increasing disease severity.

The results presented in this study after allergen challenge were all obtained 24 h after allergen instillation. They provide no evidence for any alteration to surfactant phospholipid composition that may have occurred at earlier time points. Indeed, it is possible that any such changes may have resolved by 24 h. This distinction is important given previous reports of the kinetics of secretory phospholipase A2 activation in asthmatic subjects after allergen challenge (6, 7).

Our observation of phospholipids characteristic of plasma in BALF of mild asthmatic subjects 24 h after local allergen challenge has potentially important implications for concepts relating inactivation of surfactant to the acute disease process in asthma. Phospholipids are transported in plasma as integral components of lipoproteins, which implies that lung inflammation in asthma causes sufficient disruption of airway epithelial cell junctions to permit infiltration of relatively large lipoprotein particles. This conclusion is important because lipoprotein components cannot be measured in airway or alveolar fluid from the lungs of healthy people or from many patients with a variety of chronic conditions. In addition to this lipoprotein infiltration being relatively specific to asthma, the absence of plasma phospholipid components in BALF from asthmatic subjects under baseline conditions suggests that such infiltration is a transient component of the acute disease response.

Increased concentration of protein components of plasma such as fibrin (21) has previously been suggested to contribute to the severity of the asthmatic response by direct inhibition of the surface tension properties of surfactant (8). In this model, it is envisaged that impaired surfactant function potentiates airway edema and results in liquid accumulation in the narrowest sections of conducting airways. This, in turn, could then contribute to the mucus plugging and reduced airflow characteristic of acute asthma. It is also possible that the lipoprotein component of the plasma infiltration of BALF could be an additional factor in this response. This concept is supported by a recent report (1) that described the physical interactions between serum lipoproteins and surfactant. Analysis of BALF from a patient with extensive lipoprotein infiltration of the lungs demonstrated the presence of abnormal complexes between surfactant and serum lipoproteins, probably due to the binding of surfactant protein A to lipoprotein. The formation of comparable surfactant-lipoprotein complexes might be expected to contribute to the impaired surfactant function in the acute asthmatic response.

In summary, we have shown that phospholipids characteristic of serum lipoproteins entered the airways of asthmatic subjects after local allergen challenge. Although such infiltration is not a direct cause of the asthmatic response, it might potentiate the extent of disease severity. Interactions between surfactant and lipoproteins might impair surfactant function. Finally, analysis of phospholipid molecular species in BALF provides a rapid and sensitive approach to detect airway lipoprotein infiltration in lung disease.


    ACKNOWLEDGEMENTS

We thank Prof. Peter Shoolingin Jordan, Neville Wright, Paul Skipp, and Kathy Ballard (Department of Biochemistry and Molecular Biology, University of Southampton, Southampton, UK) for access to and assistance in using the electrospray ionization mass spectrometer.


    FOOTNOTES

This work was supported by an equipment grant from the Wellcome Trust (to Prof. Shoolingin Jordan) and Project Grant Kr 1405/2-1 from Deutsche Forschungsgmeinschaft.

E. L. Heeley was supported by Wessex Medical Trust Studentship Grant 445/13.

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 and other correspondence: A. D. Postle, Child Health (803), Level G, Centre Block, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK (E-mail: adp{at}soton.ac.uk).

Received 6 April 1999; accepted in final form 20 September 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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