Platelet-activating Factor in Bronchoalveolar Lavage from Patients with Sarcoidosis1-3

ERMANNO SCAPPATICCI, DANIELA LIBERTUCCI, FLAVIA BOTTOMICCA, RAFFAELLA DA COL, WIGI SILVESTRO, CIRO TETTA, and GIOVANNI CAMUSSI

Introduction

Platelet-activating factor (PAF), initially recognized as a mediator of anaphylaxis released by antigen-stimulated IgEsensitized rabbit basophils (1), belongs to a new class of lipid chemical mediators, the acetylated alkyl phosphoglycerides (for review see reference 2). PAF exerts a broad spectrum of diverse and potent biologic activities. Besidesits activity on platelets (1),PAF promotes the aggregation, chemotaxis, and granule secretion of polymorphonuclear neutrophils (PMN), eosinophils, and monocytes (2). PAF enhances vascular permeability, leading to hemoconcentration with extravasation of plasma proteins, accumulation of leukocytes, edema, vasoconstriction, and vasodilation (2). On the basis of its multiple biologic activities relevant for the development of the inflammatory reaction, it was postulated that PAF may playa role in several physiopathologic alterations of the lung (for reviewsee reference 3). Indeed, PAF may be synthesized by lung resident cells such as mastocytes (2) and macrophages (4, 5) or by inflammatory cells recruited in the lung such as PMN, monocytes, and eosinophils (5). PAF produced locally may participate in a mediator network that amplifies inflammatory reactions (6). In addition, because specific binding sites for PAF are present in human lung tissue (7), a direct action of PAF on the lung may also be envisaged. Recently, PAF was implicated as a mediator potentially involved in the pathogenesis of asthma (3). The inhalation of PAF produces bronchoconstriction in normal and asthmatic subjects and increases airway reactivity to methacholine (2, 8). Alveolar macrophages obtained from bronchoalveolar lavage (BAL) of asthmatic patients release PAF in the presence of specific antigen (4), and eosinophils isolated from asthmatic patients release more PAF than do those obtained from nonasthmatic subjects (9).

SUMMARY Platelet-activating factor (PAF), a lipid mediator of Inflammation and anaphylaxis, may playa role In several physiopathologic alterations of the lung. A lipid compound with physicochemical and biologic characteristics similar to synthetic PAFwas extracted and purified from bronchoalveolar lavage (SAL) fluid of 15 of 34 patients with sarcoidosis. PAF was quantitated by a bioassay on washed rabbit platelets. The specificity of platelet aggregation was assessed by using two different PAF receptor antagonists. The Incidence of detectable amounts of PAF In SAL fluid of sarcoid patients was statistically significant (x 2 = 4.064, P = 0.044) when compared with the 14 normal control sUbJects.The results demonstrated an Increased production of PAF In the lower respiratory tract of patients with sarcoidosis. The presence of PAFIn SAL fluid, however, did not correlate with radiologic stage, Intensity of alveolltls, gallium scanning positivity, angiotensin-converting enzyme serum level, or lung function tests. Therefore, a direct relationship between presence of PAF In SAL fluid and activity of lung disease In patients with sarcoidosis was not directly established. AM REV RESPIR DIS 1992; 146:433-438

The presence of PAF was demonstrated both in the sputum (10)and in the BAL fluid of asthmatic patients (11). In addition, PAF may be involved in the pathogenesis of adult respiratory distress syndrome (ARDS), a disease characterized by endothelial injury and accumulation of platelets and neutrophils within pulmonary blood vessels (3). Such pathologic alterations can be mimicked by intravenous injection of PAF in experimental animals (12). On the basis of results obtained in the Arthus reaction (13), which is induced by in situ formation of immunocomplexes, it seems possible that PAF may contribute to the pathologic events of extrinsic allergic alveolitis. Finally, because pulmonary endothelial cells may produce PAF (14), a potential role of this mediator in pulmonary vascular disease has been postulated (3). PAF, in fact, may directly increase vascular permeability in the lung (14), and it may promote neutrophil-mediated endothelial damage (15). The aim of the present study was to assess the presence of PAF in the BAL of patients with pulmonary sarcoidosis. Sarcoidosis is a chronic multisystem granulomatous .disorder characterized by accumulation of activated T lymphocytes and macrophages and granuloma formation at the sites of tissue injury. In the lung the intensive inflammatory alveoli-

tis and sarcoid granuloma formation may lead to progressive interstitial fibrosis (16). The results of the present study demonstrate a local generation of PAF in the lower respiratory tract of patients with pulmonary sarcoidosis. Methods Patients and Control Subjects The study population consisted of 34 nonsmoking patients with untreated Stage I and Stage II sarcoidosis. The diagnosis of sarcoidosis was histolog-

(Received in original form April 8, 1991 and in revised form February 11, 1992) 1 From the Servizio eliFisiopatologia Respiratoria e Broncologia, Ospedale San Giovanni, Servizio di Anatomia Patologica, Dipartimento di Scienze Biomeelichee Oncologia Umana, Universita eliTorino, Respharma Pharmacological Research, Torino, and Laboratorio eliImmunopatologia, Cattedra di Nefrologia, Universita di Torino e Cattedra di Nefrologia Sperimentale, Dipartimento eliBiochimica e Biofisica, I Facolta di Medicina e Chirurgia, Napoli, Italy. 2 Supported by a grant from the Consiglio Nazionale delle Richerche: Progetto finalizzato prevenzione e controllo dei fattori di malattia (FATMA); Sottoprogetto cause di malattie da infezione (CTN920037.PF41). 3 Correspondence and requests for reprints should be addressed to Prof. G. Camussi, Laboratorio di Immunopatologia, Cattedra di Nefrologia, C. Polonia 14, Torino, Italy.

433

434 ically confirmed by the presence of noncaseating granulomas on the lung and/or lymphonode biopsy (mediastinoscopy), without clinical or biologic evidence of other causes of granuloma formation (16).All the patients weresubmitted to lung function tests, gallium67 lung scanning, angiotensin-converting enzyme (ACE) assay, and bronchoscopy for BAL and transbronchial biopsies. For comparison BAL samples from eight nonsmokers and nonatopic healthy volunteers were evaluated: they had neither history nor evidence of lung disease upon physical examination, chest radiographs, and pulmonary function tests. All subjects studied gave informed consent.

Lung Function Tests Measurement of FVC, FEV 1, and carbon monoxide single-breath diffusion capacity (OLeo) were performed using an HP-47120 spirometer (Hewlett-Packard, Waltham, MA). Percent predicted values for FVC, FEV 1, and OLeo were calculated as described by Cotes (17). Radiology A chest radiograph was available for all sarcoid patients and normal subjects at the time of BAL. Chest films were scored as Stage 0 (normal), Stage I (hilar adenopathy), Stage II (hilar adenopathy with parenchymal infiltrates), or Stage III (parenchymal infiltrates alone) (16). At the time of BAL 24 patients had radiographic Stage I sarcoidosis and 10 had Stage II sarcoidosis. Gallium Lung Scanning GaHium-67 lung scanning was performed in all patients 48 h after intravenous injection of 2 mCi of 67Gacitrate. The 67Gauptake was evaluated as "positive" if it was observed to be equal to or greater than the diffuse uptake of normal liver or "negative" if no 67Gauptake was observed (16). Angiotensin-converting Enzyme Assay The level of serum ACE was determined by a radioimmunoassay kit (Sorin Biomedica, Saluggia, Italy). Results were reported as units per liter (normal values < 150). Bronchoalveolar Lavage Bronchoscopy with analysis of BAL fluid was performed in a standard fashion as previously described (18). Briefly, the lingula or right middle lobe was washed with 180 to 200 ml of saline prewarmed to 37° C. Ten milliliters of BAL fluid were immediately processed for PAF extraction as described below, and the remaining fluid was filtered through sterile gauze and kept on ice until the cell recovery was determined by hemocytometer. The differential cell count was performed on cytocentrifuge preparations stained with WrightGiemsa. On the basis of the percentage of T-Iymphocyte recovery, patients were grouped in high-intensity alveolitis (HIA) (lymphocytes >28070) and low-intensity alveolitis (LIA) (lymphocytes < 28070) (16). In the Stage I

SCAPPATICCI, LlBERTUCCI, BOTTOMICCA, DA COL, SILVESTRO, TETTA, AND CAMUSSI

group, 11 patients had HIA sarcoidosis and 13had LIA sarcoidosis; in the Stage II group, three patients had HIA sarcoidosis and seven patients had LIA sarcoidosis. Lymphocyte subsets were furthermore defined by monoclonal antibodies LEU 3 (CD4 helper-inducer T lymphocyte) and LEU 2 (CDS suppressor-cytotoxic T lymphocyte) (Becton-Dickinson, Lincoln Park, NJ).

Purification and Characterization of PAF Ten milliliters of BAL samples were extracted and purified by methods previously described for the recovery of PAF from biologic specimens (13, 19). Briefly, immediately after recovery, 1 mg of bovine serum albumin (BSA) (Pentex Fraction V; Miles Laboratories, Kankakee, IL) was added as PAF carrier, and the samples were acidified to pH 3.0 to 3.5 with IN HCI to destroy the acid-labile inhibitor of PAF. After stirring for 10 min the cells were removed by centrifugation, the supernatant was adjusted to pH 5.5 to 6.0 with IN NaOH, and lipids were extracted by 1.9 vol of methanol. After a 1-h incubation at room temperature, the precipitated proteins were removed by centrifugation at 10,000 g for 20 min at 4° C, and 2 vol of chloroform, 0.1 vol of methanol, and 0.8 vol of water were added to the supernatant to effect phase separation (19). The lower chloroform-rich phase was then subjected to thin-layer chromatography (TLC) on precoated silica gel plates 60F254 (Merck, Darmstadt, Germany), using chloroform:methanol:water (65:35:6 vol/vol/ vol) (19) as the solvent system. Subsequently, each plate was sectioned into l-cm segments starting at the origin, and the lipid of each fraction was extracted according to the method of Pinckard and coworkers (19) and further characterized by its retention time on a high-performance liquid chromatography apparatus (HPLC) (Beckman Instruments, Palo Alto, CAl equipped with a J.l,Porasil column (Millipore Chromatographic Div., Waters, Milford, MA). Elution was carried out with chlorcform:methanol:water, (60:55:5 vol/vol/ vol) at a flow rate of 1.0 ml/min. Synthetic PAF (Bachem Feinchemikalien AG, Bubendorf, Switzerland), sphingomyelin, and lyso2-phosphatidylcholine (Lyso-PC) (Sigma, St. Louis, MO) were used as reference lipids. The fractions were dried under N 2 , resuspended in 0.5 ml TRIS-buffered Tyrode's solution with 0.25070 BSA, and bioassayed on washed rabbit platelets as described below. The recovery of synthetic PAF was evaluated by mixing 15 ng of synthetic PAF (1-0exadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (Bachem Feinchemikalien AG) or 0.5 J.l,Ci of [3H]PAF (120 Ci/mmol) with 10 ml of BAL from a normal subject, followed by extraction, TLC, and HPLC. The recovery of synthetic [3H]PAF was evaluated as radioactivity (range, 96 to 100070). All samples eluted from the HPLC column and active on washed rabbit platelets were characterized as containing PAF by comparison with synthetic PAF and with PAF derived from IgE-sensitized rabbit basophils (19) according to the following criteria.

(1) Chromatographic pattern on TLC using chloroform:methanol:water (65:35:4 vol/vol/ vol or 65:35:6 vol/vol/vol) as a solvent, with migration between Iyso-PC and sphingomyelin (2). (2) Physicochemical characteristics such as inactivation by strong bases, resistance to acid and weak basic conditions (19), and inactivation by phospholipase A 2 but not phospholipase AI' The methods used have been described in detail in previous reports (13).Briefly,base-catalyzed methanolysis was performed in 2 ml 0.03N NaOH dissolved in methanol with an incubation of 5 min at 22° C, followed by acidification of the samples to pH 6.5 ± 0.1 with 3N HCI. PAF was subjected to acidic treatment for 3 h at 22° C; the samples were dissolved in 1 ml chloroform:methanol (9:1 vol/vol) to which 1 vol 0.03N HCI . was added. To test the effect of weak basic conditions, PAF dissolved in 1 ml chloroform:methanol (9:1vol/vol) was reacted with 0.5 vol ammonium hydroxide 28070 for 30 min at 22°C under stirring, followed by acidification to pH 6.5 ± 0.1 with IN HCI. At the end of the various chemical treatments, the lipids were extracted according to the method of Bligh and Dyer (20). The treatment with different lipases was performed on dried samples. The assay mixture for phospholipase A 2 contained 1 ml TRIS-buffered 150mM NaCI at pH 8.0 with 10 mM CaCho Before the addition of 0.03 mg phospholipase A 2 , the mixture was sonicated (100 W for 5 min in an ice bath). The reaction was performed at 37° C for 1 h. Lipase At hydrolysis was accomplished by sonicating the sample (100 W for 5 min in an ice bath) in 1 ml borate buffer 0.1 M at pH 6.5 with 10 mM CaCI2 , 1 mM deoxycholate, and 0.4070 BSA, then by incubating the mixture with 0.2 mg of the enzyme for 22 h under stirring. After the lipase treatment, the lipids were extracted according to the method of Bligh and Dyer (20). In control experiments, treatment of [3H]PAF (0.5 J.l,Ci) with phospholipase A 2 or basecatalyzed methanolysis hydrolyzed 94 to 99070 of PH]PAF to lyso-PAF. Lipase At as well as acidic and weak base conditions did not cause significant hydrolysis of [3H]PAF (zero to 5070). After base-catalyzed methanolysis or phospholipase A 2 treatment, lipids were acetylated in the presence of 1 ml of acetic anhydride and 1 ml pyridine for 20 h at room temperature (21). After lipid extraction as described above, the biologically active material was tested on washed rabbit platelets and compared with untreated control material. (3) Ability to induce platelet aggregation independently from both ADP and arachidonic acid thromboxane-As-mediated pathways (13). The specificity of platelet aggregation was inferred from the inhibitory effect of platelet pretreatment (30 s at 37° C under continuous stirring at 900 rpm) with 5 J.l,M of SRI 63072 (Sandoz Research Institute, East Hanover, NJ) and ofCV-3988 (Takeda Chemical Industries, Kyoto, Japan), both PAF receptor antagonists. (4) In four samples containing 1.700 to 3.200pg/ml of PAF-likebioactivity, the chem-

435

PAF AND WNG SARCOIDOSIS

ical identity with the synthetic PAF (1-0exadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was evaluated by anewly developed technique based on high pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) (Silvestro and colleagues, unpublished observations). An API III mass spectrometer (Perkin-Elmer SCIEX; PerkinElmer Medical Instruments, Oakbrook, IL) with an ionspray articulated source, interfaced to a syringe pump HPLC (No. 140A;Applied Biosystems, Foster City, CAl was used. Briefly, each sample purified by TLC was resuspended in 30 ul of tetrapropylammoniuml formiate 3.3 mM at pH 4.0 and acetonitrile (70:30 vol:vol), and 20 ul of sample were injected in HPLC on a reverse-phase butyl widepore column (Hypersil WP-Butyl 100 x 1 mm; Shandon Southern Instruments, Sewickley, PAl using a mobile phase gradient composed of (A) acetonitrile/tetrapropylammonium 33 mM in water brought to pH 4.0 with formic acid (90:10), and (B) tetrapropylammonium 3.3 mM in water brought to pH 4.0 with formic acid. After 5 min ofisocratic elu- ~ tion at 300/0 of A, the concentration of A was increased to 1000/0 in 20 min with a linear gradient. The flow rate was 50 ul/min; a postcolumn makeup of 30/0 trifluoracetic acid at 5 ul/min (Harvard syringe pump; Harvard Apparatus Co., South Natick, MA) was used to improve the ionization. Mass spectrometry analyses were performed under MS/MS conditions. Daughter ions, in the range 40 to 560 m/z, wereacquired in positive mode from parents of 524 m/z, which correspond to the protonated molecular ion of CI6-PAF. Fragmentation was obtained by collision with argon at a collision

gas thickness of 4 x 1012 atoms/em and at an impact energy of 70 eVe Standard CI6-PAF was purified and analyzed with the same technique.

PAF Assay After extraction and purification by TLC and HPLC, PAF was quantitated by aggregation of washed rabbit platelets in the presence of 10J,lM indomethacin (Sigma), which inhibits cyclooxygenase, and of creatine phosphate (312.5 ug/mlj/creatinine phosphokinase (152.5 ug/ml) enzymatic system (Sigma), which converts ADP to ATP, thus blocking the arachidonic acid- and ADP-dependent platelet aggregation, respectively. The amount of PAF was expressed in picograms per milliliter and calculated over a calibration curve of synthetic PAF constructed for each test. Statistical Analysis The data were evaluated by Student's t test and by the "I} test (22). Values were given as means ± SD; p values < 0.05 were considered statistically significant. Results

Sarcoidosis was classified as Stage I or Stage lIon the basis of radiographic findings and as high intensity or low intensity alveolitis on the basis of the percentage of lymphocyte recovery from BAL (table 1). The values of FVC and Dteo, the percentage of macro phages and lymphocytes recovered from BAL, the serum level of ACE, and the incidence of gallium-67 lung scanning positivity in the

~F ml 3500

0

2800

0

8 C' C

2100 1400

0 0 C

700

0

NORMALS I HIA

ILIA

Stage II Sarcoidosis L1A

Number Vital capacity, % predt Diffusing capacity, % pred t BAL macrophages, 0/0 BAL lymphocytes, 0/0 ACE, UIL Gallium scan positivity

11 104.18 89.27 54.36 41.82 197.09

± 8.12 ± 16.62 ± 7.8 ± 7.25 ± 42.12 10/1

HIA

13 94.85 88.46 86.31 10.46 139.77

± 12.77 ± 13.42 ± 7.76 ± 7.98 ± 43.67 815

L1A

± ± ± ± ±

Normal Subjects

7

3 100 91.33 59.33 36 176.43

6.29 17.62 16.77 12.17 87.66

2/1

91.3 70.29 81 15.57 160.67

± 21.63 ± 22.43 ± 6.27 ± 5.35 ± 36.46 5/2

14 95.71 96.21 91.62 6 98.5

± 8.42 ± 9.45 ± 6 ± 3.3 ± 24.66 NO

Definition of abbreviations: HIA • high intensityalveolltis; L1A • low Intensityalveolitis;ACE. angiotensin-converting enzyme;NO • not determined. • Data are given as mean :t SO. t For reference values, see reference 16.

TABLE 2 PAF IN BAL FLUID FROM PATIENTS WITH SARCOIDOSIS AND NORMAL SUBJECTS

Patients, n Patients with PAF, n Median PAF concentration, pglml Range, pglml • Data are given as mean :t SO.

Sarcoidosis

Normal

,34 15 700 ± 1,040* 0-3,200

14 0 0 0

X2

IILIA

different groups of patients with sarcoidosis and in the normal subjects are also listed in table 1. PAF was detected in BAL fluid from 15 of the 34 patients studied, but not in the fluid of the normal subjects (Xl = 4.064; p = 0.044) (table 2). The distribution of PAF levels among the patients classified on the basis of radiologic findings and intensity of alveolitis is shown in figure 1. Patients with and without PAF in BAL fluid were compared (table 3). No significant differences were found concerning age, sex, radiologic stage, total BAL cellularity, intensity of alveolitis, T4/T8 ratio and percentage of macrophages on BAL fluid, serum level of ACE, or gallium-67 lung scanning positivity. PAF present in BAL fluid had phys-

TABLE 1

HIA

IIHIA

Fig. 1. Distribution of PAFlevels in SAL fluid of patients with sarcoidosis and that of normal subjects.

LABORATORY DATA IN PATIENTS WITH SARCOIDOSIS AND IN NORMAL SUBJECTS· Stage I Sarcoidosis

0

00

0

0

t Test

p Value

2.502

0.044 0.016

4.064

436

SCAPPATICCI, LlBERTUCCI, BOTTOMICCA, DA COL, SILVESTRO, TETTA, AND CAMUSSI

TABLE 3

TABLE 5

COMPARISON OF PATIENTS WITH SARCOIDOSIS WITH AND WITHOUT PAF IN BAL FLUID

EFFECT OF PAF RECEPTOR ANTAGONISTS ON WASHED RABBIT PLATELETS AGGREGATION

Sarcoidosis with PAF

Sarcoidosis without PAF

15 21-51 7/8 12

19 24-71 3/16 12

Patients, n Age. range Sex. M/F Stage I Stage II HIA Cells. number/ml * t TiT. ratio* BAL macrophages. % * ACE. U/L* Gallium-67 scan positivity

14.64 6.41 72.53 184.33

± ± ± ±

0.506 0.421 0.109 0.489 0.522 0.812 0.813 0.332 0.632 0.633 0.137 0.69

7 7

3 7 8

LlA

p Value*

12.61 5.38 69.84 154.58

4.45 x 1()4 5.41t 16.5t 71t 12

12 ± 3.91 x 104 ± 6.7 ± 18.65 ± 41.98 13

CV 3988. 5 ~M SRI 63072. 5 ~M

TABLE 4

Synthetic PAF

0.22 0.10

0.22 0.10

20

20

o

o

0-6

0-5

Definition of abbreviations: TLC = thin-layer chromatography; HPLC matography; Rf = retardation factor; Rt = retention time. * Percent recovered activity.

91-96* 100

90-98 100

aggregation of washed rabbit platelets by an ADP- and arachidonate-independent pathway. This aggregation was inhibited by two different PAF receptor antagonists, SRI 63072 (23) and CV 3988 (24) (table 5). The MS/MS spectra obtained from BAL samples at the retention time characteristic for C-16PAF (13.4to 13.7min) exhibited a fragmentation pattern characterized by a molecular ion (m/z 523) and a fragment corresponding to phosphocholine (m/z 183). Figure 2A shows a typical spectrum of a BAL sample containing 3,200 pg/ml of PAF-like bioactivity. An identical spectrum was obtained with synthetic C-16 PAF, either submitted to the same extraction and purification procedures of the samples (figure 2B)or injected as such (figure 2C).

PHYSICOCHEMICAL CHARACTERISTICS OF PAF BIOACTIVE MATERIAL EXTRACTED FROM SPECIMENS

TLC Solvent system CHCI:s:CH:sOH :H2 0 65:35:6. Rf 65:35:4. Rf HPLC Rt. min Base-catalyzed methanolysis * Phospholipase A2 treatment*

Synthetic PAF

* Percent inhibition of platelet aggregation.

For definition of abbreviations, see table 1. * Data are given as mean ± SO. t Cell number per milliliter in normal sUbjects = 6.8 ± 2.9 x 104.

PAF-Iike BAL Material

PAF-Iike BAL Material

= high performance liquid chro-

Discussion

icochemical and biologic characteristics similar to those of PAF released from IgE-sensitized rabbit basophils and of synthetic PAF (tables 4 and 5). (1) It had the same migratory properties on TLC (Rr = 0.22, with a solvent system chloroform:methanol:water ratio of 65:35:6 vol/vol/vol) between lyso-PC (Rr = 0.11) and sphingomyelin (Rr = 0.29) (19). No PAF activity was detected in other TLC fractions. Using a solvent system with different pH (neutral, acid, and basic), the Rr of PAF extracted from BAL fluid was similar to synthetic PAF, indicating its neutral nature. However, using a solvent system with different polarity (chloroform:methanol:water, 65:35:4 vol/ vol/vol), the Rr of PAF present in the BAL as wellas that of synthetic PAF were significantly changed (Rr = 0.10), indicating the polar lipid nature of the biologically active material extracted (19). By HPLC, PAF present in the BAL coeluted with synthetic PAF with a retention time (Rt ) of 20 min. (2) PAF activity was destroyed after base-catalyzed methanolysis or after treatment with

In the present study, a lipid compound with physicochemical and biologic characteristics similar to 1-0-exadecyl-2acetyl-sn-glyceryl-3-phosphorylcholine (PAF) was extracted in significant amounts from cell-free BAL fluid in 15 of 34 patients with sarcoidosis. The demonstration that PAF is present in BAL fluid of some sarcoid patients, but not in normal subjects, suggests a potential role for this mediator in the development of inflammatory reactions

phospholipase A 2 • The treatment with lipase At did not inhibit the PAF activity, suggesting the presence of an ether bond at sn-l. Acidic condition had no effect on PAF activity. Treatment with a weak base suggested that the biologically active material did not contain ionizable groups. After base-catalyzed methanolysis or inactivation with phospholipase A 2 , treatment with acetic anhydride restored the biologic activity. (3) It induced

A

fLJ 19 1

183.18

100

L

13.6

J

523.52

!·~~l o.O_lI4IItlP:l*IIlIi"¥.l..yr~ '::~~~gqgu~~~~~~"",-~~ .•

i:: t]C

1ef

~I

200

: 1O.1~ 51 400

mlz

Fig. 2. Spectra of the daughter ions from parents with m/z 524 of a BAL sample (A). C16-PAFsubmitted to the same extraction and purification process of BAL samples (B),and synthetic C16-PAF as such (C). In the inserts. reconstructed chromatograms of the daughter ions with m/z 183 from parents of m/z 524 of the correspondingsamplesare shown. Fragmentation spectra are identical in A, B, and C. Only one peak can be observed in each ch romatogram with superimposable retention time.

PAF AND WNG SARCOIDOSIS

that characterize lung sarcoidosis. However, we failed to establish a positive correlation between the presence of PAF in BAL fluid and radiographic stages or intensity of alveolitis and other markers of disease activity. A similar discrepancy between presence of PAF in BAL fluid and activity of the disease has been reported in asthma (11). Several technical problems may interfere with the recovery and quantitation of PAF present in BAL fluid, even if lipid extraction is performed immediately in order to avoid the reported loss of PAF from stored BAL fluid (25). It is conceivable that the amount of PAF generated may be underestimated because of the large dilution of fluids inherent to the BAL technique itself, its binding to the surface of cells, or its inactivation by specific acid-labile acethylhydrolases present in plasma and in many," cells (26). This enzyme converts PAF in- ' to 2-lyso-PAF by removing the acetyl group from Position 2. In the present study, 1070 BSA was added to BAL samples immediately after recovery to provide a critical concentration of proteins as a PAF carrier because of its relative insolubility in aqueous media. In addition, in order to avoid inactivation of PAF by acetylhydrolase, the BAL samples were acidified immediately after recovery, before removing cells by centrifugation. Despite these cautions we cannot exclude that a significant catabolism had already occurred in the peripheral airways. In fact, in isolated perfused rat lung, PAF is rapidly and extensively metabolized into long chain 2-acyl-glyceryl-phosphorylcholine molecular species, mainly by alveolar type II cells, non ciliated bronchiolar epithelial cells, and fibroblasts (27, 28). This metabolic pathway may reflect an inactivation mechanism to protect lung from PAF biologic activities (27). It was recently reported that intracellular levelsof PAF may be relevant in the pathophysiologic behavior of inflammatory cells (29). The number of cells recovered by centrifugation of the 10-ml BAL samples used for PAF extraction was insufficient for measuring PAF intracellular levelsin patients with sarcoidosis. Despite the technical difficulties involved in studying the role of PAF in the development of a localized inflammatory process, its detection in the lower respiratory tract of 15 of 34 patients with a lung interstitial inflammatory disease such as sarcoidosis may suggest an increased local production of this mediator.

437

The potential role of PAF in inducing interstitiallung injury has been established in experiments of PAF instillation into the airways of rabbits (30). PAF causes a dose-dependent inflammatory reaction characterized by an accumulation of neutrophils and platelets in the alveolar capillary lumina and of macrophages, eosinophils, and a minor number of neutrophils in septa and in alveolar spaces. Macrophages accumulated in the interstitium and in the alveoli had morphologic aspects of activation with large numbers of pseudopods, phagocytosis of injured epithelial cells, and degranulation. These alterations, which occurred a few hours after a single instillation of PAF, were followed by the development of pulmonary fibrosis (30). Similar alterations with a prominent accumulation of platelets in the pulmonary interstitium were observed in guinea pigs (31). In baboons, PAF instillation caused a prominent accumulation of eosinophils in the alveoli that persisted for as long as 2 wk (32). In normal and allergicsubjects, PAF inhalation was associated with accumulation of leukocytes, mainly neutrophils, in BAL fluid, suggesting that PAF mediates recruitment of inflammatory cells in human lungs also (33). PAF is often only one of many molecules capable of similar effects in inflammation (6). PAF may stimulate the production of new mediators from its target cells;thus, it is difficult to ascribe to PAF a specific effect in the complex interplay of the inflammatory reaction. The presence or absence of a given mediator, including PAF, in BAL fluid from patients with lung allergic or inflammatory reactions cannot be taken as direct evidence for or against its involvement. This would require concomitant information on its inactivation, clearance, intracellular metabolism, and recovery. Even then it would be difficult to identify the precise pathogenic role of PAF in vivo. In conclusion, we have demonstrated that PAF is present in BAL fluid of patients with sarcoidosis. The failure to establish a correlation with the activity of the disease, possibly because of technical problems or the timing of BAL sampiing, does not allow us to establish a pathogenetic correlation with the development of lung injury. Further studies are needed to ascertain whether the presence of PAF in the lower respiratory tract of patients with sarcoidosis is indicative of a pathogenic role or is a mere epiphenomenon of an ongoing inflammatory process.

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