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Mechanisms of damage to the lung surfactant system
:XPERIMENTbL
AND
MOLECULAR
Mechanisms
of Damage
Ultrastructure
14,
PATHOLOGY
to the
and Quantitation inactivated
JOHN
Department
U.
BALIS,
o/Pathology, Maywood,
243-262
(1971)
Lung
Surfactant
of Normal
Loyola
of Chicago,
University
60153 und V.A. ZZeceivecl
and In Vifro
Lung Surfactant
SUE A. SHELLEY, MILES EDWARD S. RAPPAPORT
Illinois
System’
Hospital,
Decembe?
J.
MCCUE,
StTitch Bines,
AND
School
Illinois
of Medicine,
GOlgi
15,19iO
This study provides a structural, functional and biochemical basis for the characterization and quantitation of normal and in vitro inactivated lung surfactant. 1. Normal lung surfactant. Rabbit lung washing was centrifuged for 5 minutes at 4509 to obtain the cellular sediment A. The supernatant was centrifuged for 90 minutes at 100,OOOg yielding sediment B and supernatant C. All fractions were lyophilized and weighed. In addition, sediments A and B were processed for histochemistry and electron microscopy. Surface tension measurements revealed minimum surface tension (7 min) of O-5 dynes/cm with 5 mg of lung washing or with 2 mg of sediment B. By contrast, even with 20 mg of sediment A or supernatant C, y min was above 18 dynes/cm. By electron microscopy sediment B was composed mainly of myeloid figures. Biochemical determinations of lyophilized fractions revealed that more than 80% of the noncellular phospholipids present in the bulk sedimented into the strongly surface active sediment B, which was found to contain an average of 721 pg phospholipids/mg dry weight, with 90% of the phospholipid being phosphatidyl choline. Fractions A and C revealed relatively low phospholipid values, 214 pg/mg dry weight and 129 pg/mg dry weight, respectively. 2. In vitro inactivation of lung surfactant. (a) Interaction of lung washing with serum resulted in a seemingly reversible form of surfactant inactivation, since sediment B of the surface inactive washing-serum mixture revealed a high phospholipid content and a y min of O-5 dynes/cm with 2 mg of lyophilized material. (b) Following interaction of lung washing with heparinized or recalcified titrated plasma, a clot rapidly developed in the washing-plasma mixture due to the strong thromboplastic activity of surfactant. By electron microscopy the clot contained in addition to fibrinous deposits. packed macrophages, and numerous myeloid figures. Similar changes but no cells were observed in the clot formed in a mixture of cell-free surfactant suspension with plasma. Lipid analysis and isotopic tracing studies indicated that the clot incorporated 50-60% of the surfactant phopsholipids present in the plasma inactivated lung extracts. On the other hand the eluate of the washing-plasma mixture yielded a sediment B with low phospholipid content and y min of 20-25 dynes/cm. This form of surfactant inactivation can be defined, therefore, as a coagulative type of surfactant depletion, and may represent the mechanism of surfactant deficiency and membrane formation in hyaline membrane disease of the newborn and in other pathologic conditions of the lung associated with formation of hyaline membranes. 1 Study supported Health Service Grant
in part by HD-O4434-01.
USAMRDC
Contract 243
DADA
17-70-0041
and
U.S.
Public
244
lLU,TS E2’ AL.
Lung surfactant, is a complex? noncellular, surface active material which coats the alveoli of the normal lung (hvcry and Said, 1965; Pattle, 1965; Clements and Tierney, 1965; Scarpelli, 1968). The surface active component of the lung surfactant is a mixture of pl~ospholipitls:, hountl to proteins and carbollydrntes, and its main fuctioii is to reduce surface tension and stabilize air spaces. Hyalinc membrant~ discasc of the newborn is the most important clinicopathologic entity with an unclerlying surfactant defrct (Avery and Said, 1965). Lung surfactant is also d:maged in shock (Henry! 1968; Mart,in et nl., 1968)) pulmonary embolism (Greenfield and Duncan, 1968; Wolfe and Sabiston, 1968)) oxygen toxicity (Giammona et rrl., 1965) and in other pat,hologic conditions of the lung characterized by atrlccta. *‘u, 1, edema, h(~morrhages,and often formation of hyaline membranes in alveolar spaces. The pathogenctic interrelationships bct’ween the above lung changes and surfactant deficiency remains uncertain, in spite of extensive work in this field. From various lines of evidence it’ appears that the lung surfactant is synt’hesizcd and aecrctcd by the type II epithelial cells (Macklin, 1954; Klaus et ul., 1962; Buckingham et nl., 1966; Balis and Concn, 1964; Bensch et (11.,1964; Kikkawa et (11.,1965; Sorokin, 1966; Kikkawa et al., 1968; Kuhn, 1968)) but primary surfactant deficiency due to impaired synthesis of surfactant has never been convincingly demonstrated. On the other hand, recent studies appear to support the concept that interaction of t,he lung surfact’ant with blood constituents may lead to damage or inactivation of lung surfactant (Tnylor and Ahrams, 1966). T_;nfort~uuatcly, at, present. it is not’ possible to clefinc and differentiate distinct forms of aurfnctant, tlamngc or inactivation. This is due to the fact that the structural fcaturcs of normal and altered surfnctant hare not been correlated wit,11quantitative, functional, and biochemical parameters relating to surfactant activity or deficiency. From studies in our laboratory, methods have been clrvclol~tl which provide the opportunity to (a ) identify, recover, and quantitate even minute amounts of surface active fractions from lung extracts! and (b) meaningfully correlate morl)hologic charnctt%tics of hurfac;:\nt’ components with quantitative surface tension measurements and biochemical data. Furt,hcrmore, using the above parnmctcrs and an i,,. zbitro model (Balis ant1 Rappaport, 1969; Balis et (II., 1970), it has been possible to study and characterize reversible and irreversible forms of surfactant inactivation following intcraction of blood constituents with lung surfactant.
Preparation of lung washing and its centrifugation fractions. Thirty-five malt albino rabbits weighing I .4-1.6 kg were ancAlctizcd by intravenous injection of sodium pentobarbital. Thr animals wcrc cx.sanguinated by cutting the aorta nncl inferior vena cara, and before opening the chest, cavity, the trachea was clwm1~~l to prevent the entry of blood into the tracheobronchial tree. Following removal of the lungs, Ringer’s lactate solution (25 ml/kg body weight) was introducccl into the lungs through a cannula in the trachea. The buffer solution was instilled over a period of 1 minute, was allowl-cclto remain in the lungs for 30 seconds, ant1 wa8 removed by gravity drainage and gcnt’le pressure. This procedure was repeated and from a total of about 70 ml of buffer solution introduced into the
NORMAL
AND INACTIVATED
LUNG
SURFACTANT
245
lungs 48-50 ml of white, opalescent washing was recovered. Ringer’s lactate rather than isotonic saline was used for the alveolar lavage because pilot studies indicated that saline results in significant damage of the alveolar macrophages. The washing was filtered through a four-layered gauze to remove mucus, and it is designated herein as bulk. A 5-ml sample of the bulk was placed separately and the remainder was centrifuged at 450g for 5 minutes to obtain the cellular sediment A. A cell count of the bulk and the supernatant obtained following the above initial centrifugation revealed an average of 50,000 and 200 cells per mm3, respectively. Thus, more than 99% of the cells present in the bulk sedimented into the fraction A. The cell-depleted supernatant was further centrifuged at 100,OOOgfor 90 minutes at 4°C to obtain the sediment B and the final supernatant C. In some experiments, the bulk was initially centrifuged at 50009, 4°C for 60 minutes prior to ultracentrifugation in order to obtain a cell-free sediment B. Samples of 5 ml from the bulk and the supernatant C were lyophilized. The sediments A and B were also lyophilized following resuspension in 5 ml Ringer’s lactate. All above fractions were placed prior to lyophilization in dehumidified and preweighed 10 ml bottles. The dry weight of the lyophilized fraction was carefully determined by subtracting the initial weight of the bottles from their final weight. Since all fractions were suspended in Ringer’s lactate before lyophilization, the dry weight of their buffer content was determined and subtracted from the final dry weight of the lyophilized fractions. This determination was possible in view of the fact that (a) the dry weight of Ringer’s lactate was found to be constant, 9.8 + .Ol mg/ml, and (b) the concentration of sodium in all Ringer’s suspendedfractions was identical (136 f 2 mEq/liter) . Thromboplastic activity of lung extracts. Citrated rabbit plasma was mixed with the following lung extracts, the minimum surface tension (v nzin) of which was previously determined using 2-3 mg of lyophilized materials: (1) bulk (y ,min = 5-10 dynes/cm), (2) cell-free sediment B (7 min = O-5 dynes/cm), (3) supernatant C (7 min = 20-21 dynes/cm), (4) cellular sediment A (y min = 18-19 dynes/cm), and (5) lung washing filtered with Seitz filters having 1~ pores (y min = 22-24 dynes/cm). The mixtures of the above extracts with plasma were tested for surface activity. In addition, the clotting time of the mixtures of recalcified titrated plasma with extracts l-5 was determined under standardized conditions using a fibrometer precision coagulation timer. The clotting time of these mixtures was compared with the conventional Quick one stage prothrombin time of rabbit plasma in order to evaluate the thromboplastic activity of each of the above lung extracts. In vitro models. In one set of experiments, rabbit lung washing was thoroughly mixed with serum or titrated plasma in a ratio of 5:1, and from these mixtures fractions A, B, and C were obtained using the technique of differential sedimentation. Cell counts revealed that sediment A contained more than 99% of the cells present in the washing-serum mixture. In a second series of experiments, lung washing or resuspended cell-free sediment B of lung washing was mixed with heparinized plasma in a ratio of 5:l. Mixtures of the above lung extracts with titrated, recalcified plasma were also used. Following clot formation, the eluate was fractionated as the washing-serum mixture.
246
BrZI,IS
B2’
AI,.
The technique of differential sedimentation used in the in zlitro models was the same as that employed for the normal lung washing, with the exception that sediment B was repeatedly resuspended in Ringer’s lactate solution and ccnt,rifuged in order to eliminate cont’aminating serum constituents. In some cxperimerits, the pl~os1~l~olil~ids of lung cxtrants were previously labeled by intravenous injection of rabbits, 20 hours prior to sacrifice, with tritiated sodium acetate (specific activity of 2.66 Ci/mnl), 250 &i/kg body weight. These isotopic studies were performed to evaluate and compare the distribution of radioactively labeled phospholipids in (a) the ccntrifugation fractions of normal lung washing and washing-serum mixture and (b) the clot and centrifugat,ion fractions of the eluate obtained following interaction of plasma with surface active lung extracts. Electron ?nicroscopy. Fragments of clot’ and nonlyophilizcd sediments A and B, obtained respectively by centrifugation of bulk at 450g for 5 minutes and 100,OOOgfor 90 minutes, were fixed in 1% buffered osmium tetroxicle with and without prior fixation in 256 buffered glut~araldehydc. Following dehydration in ascending concentrations of ethanol solution, the sediments were embeddctl in Epon 812. Ultrathin sections were examined with an RC;2 EMU-SF electron microscope after staining wit’h uranyl acetate and lead cit’rate. In addition, 1-p thick sections were stained with toluidine blue for light microscopy. Histochemist~y. Sediments A and B as well as lung samples obt’ained before and after lavage were fixed in 10% buffered formalin with 2% calcium acetate. Paraffin embedded sections were stained with the periodic-acid Schiff reaction (PAS), diastasc-P’ad, and Halt colloidal iron as modified by Xowry. To idcntify sialomucins sections were digested with I’ibrio cholerne sialidase (General Biochemicals, Inc., Chagrin Falls, Ohio) as described by Luke and Spicer (1965). In some sections blocking by methylation was pcrformcd and the sections were stained with Mowry’s colloidal iron before and after saponification according to the method of Spicer and Lillic (1959). Resuspended sediments A and B were dried on slides, and following formalin fixation and dehydration were stained using the same histochemical procedure described abore for paraffin sections. In addition, dried sediments were treated following fixation and dehydration with chloroforni-methanol (2: 1) for 16 hours to extract the lipids, and stained with PAS and Mowry’s colloidal iron. Test for surfactant function. A Greenfield pneumatic surfactometer was used for the surface tension studies. Liquid bulk was cxamincd as described by Grccsnfield and Duncan (1968). Lyophilized materials were spread on the surface of Ringer’s solution in the trough, and for each fraction the minimum surfarc tension (y ~min) was determined with 2-20 mg of lyophilized material. Each sample was allowed to cycle repeatedly at’ 3 minute cycle times until it’ showed n stable replication of the hysteresis loop. Biochemical annly~sis. Tot,al lipid s were cstractod from the, lyopliilized fractions with chloroform-met~hanol by the method of Folch et rrl. (1957). The extracted lipids were dissolved in a known volume of chloroform-methanol and measured aliquots were used for separation of lipid classes, separation of t,he phospholipids, and total phosphorus determinations. Lipid classeswere scparatccl on silica gel G plates (Analtech, Inc.). The Polvent was composed of petroleum
NORMAL
AND
INACTIVATED
LUNG
247
SURFACTANT
ether, ether, and acetic acid (80:20:1) and the plates were developed for 7 to 8 cm. The lipids were visualized by spraying with 10% phosphomolybdic acid in ethanol followed by heating at 80°C for 5 minutes. Phospholipids were separated by two-dimensional chromatography on Whatman SG-81 silica gel-loaded paper as described by Wuthier (1966). Development in the first direction was with a solvent composed of chloroform, methanol, diisobutylketone, acetic acid, and water (90:30:60:40:8). After drying, the paper was developed in the second direction with chloroform, methanol, diisobutylketone, pyridine, and .5 M, pH 10.4, ammonium chloride buffer (60: 35: 50: 70: 12). For phosphorus determinations of the phospholipid spots, the dried papers were stained with Rhodamine 6G and examined under UV light. Total lipid phosphorus and phosphorus in each of the separated phospholipids were measured by a modification of the method of Martin and Doty (1949) as described by Wuthier (1966). The phospholipid content was calculated assuming that phospholipids contained 4% phosphorus. Total protein was measured by the method of Lowry et al. (1951). Radioactivity was measured using a Tri-Carb scintillation counter. RESULTS A. Normal
lung surfactant
Histochemical and electron microscopic findings. By light microscopy the surfact active sediment B appeared as a granular material intermixed with occasional cells. The granular material was positive (red) with PAS, and blue with the Mowry’s colloidal iron stain. The Mowry’s staining reaction could be blocked by methylation, but it was restored with saponification. Sialidase digestion also blocked the staining but pretreatment of slides with chloroform-methanol had no effect on either PAS or Mowry’s colloidal iron (Table I). In the surface inactive cellular sediment A, no extracellular elements wit,h the above histochemical characteristics could be detected. In lung tissues obtained before and after alveolar lavage, staining of sections with Mowry’s colloidal iron failed to demonstrate any alteration of the mutinous coat which is know to cover the air passages (see Discussion). By electron microscopy, the granular material seen in sediment B was found to be composed of osmiophilic myeloid figures, in association with some moderately TABLE ST.ZINING
RKACTIONS
I
OF SEDIMKNTS
FROM
LUNG
WASHING
Mowry’s ‘As
Sediment A 1. Cells 2. Extracellular spaces Sediment, B (surfact,ant)
Mowry’s
2iy;
+ -
+ -
+ -
+
+
-
Methylation
-
PAS ChloroformMethan01
tion
ChloroformMeth-
+ -
+ -
+ -
+
+
+
‘:?a:-
an01
248
BSJdS ET AL.
dense amorphous material, and occasional fragments of cells. Thcbmyeloid figures appeared as either m-horl-like membranous profiles or as irregularly shaped granules or globular bodies. The latter structures rcrcaled a characteristic orderly pattern in forms of periodic lamellac or square lattice, depending on the plane of ,qcction (Figs. l-3). Cell-free scdimcnt B revealed similar ultra~tructural fcaturca with the exception that ccl1 fragmcntjs were virt’ually absent. Swf.nce tension. ttlecrsuwttlents. Previous stutlics have demoiist~rateclthat surfactant readily sediments with ccutrifugation (Finley et crl., 1968; Cavagna et al., 1967; Galdston et al., 1969; Said et al., 19681. The results of the present8 work, however, indicate that with the initial ccntrifugation of the bulk at 4509 for 5 minutes, very little surfactant is lost in t,he cellular sediment A since the minimum surface tension (7 tnin) of this fraction is clevatcd (above 18 dynes/ cm) with 2, 5, and cvcn 20 mg of lyophilized material (Table II). Similarly, the final supcrnatant C is surface inactive with 7 ,?zin above 20 dynes/cm. By contrast, lyophilized material from sediment B obtained following ccntrifugat’ion at 100,OOOgfor 90 minutes was strongly surface active, lowering surface tension below 5’ dynes/cm wit#h 2 mg of lyophilized material containing an average of 500 pg of surface active material. Thcsc findings suggcbstthat most of the surfactant present in the bulk can bc rccorc~recl in the s:cdiment B. It al~oulcl be mcntionccl that the presence of some cells in sediment B was insignificant in terms of surface tension, since following complctc removal of t’hese cells there was no tletectablc increase in the surface activity of the xbovc scdimcnt. Further studies revealed that while the surfarc act)ivity of the bulk diluted in various proportions with Ringer’ s was rcclucecl proportionally to the degree of dilution, the sediment B obtained from the above diluted surfactant suspension was strongly surface act)ive, lowering surface tension below 5 dynes/cm with 2 mg of lyophilized material. This suggests that’ the surface act’ivity of sediment B is constant’ on a dry w(>ight#basis provided that adequate quant,itics of sedimentable surfactant are present in the initial bulk. Biochemical determinations. The above findings correlated well with the pho~pholipid determinations, which yielded consistent and readily reproducible range of values for the bulk and its ccntrifugation fractions (Table II). The rrsult:: indicated that with the technique of cliffcrent,ial sedimentation employed herein, 17% and 70% of the phospholipids present, in the bulk wcrc setliment8edinto the fractions A and B, respectively. Since the cellular fraction iz contains relatively little surfactant, as judged by the surface tension measurements and the elec,tron microscopic findings (see below), it is reasonable to assumethat the phospholipid content of this fraction only reflect s its cellular origin. From the remaining phospholipids of the bulk, therefore, more than 80% was rccovcrccl in the surface active, cell depleted sediment B. The latter fraction also revealed the highest values of phospholipids per milligram of dry weight, and these values did not increase further when cell-free sediment B was used. The results of two-climcxsional chromatography indicated that in either cell-fret or cell clepletccl scdimcnt B, phosphaticlyl choline represented 90% of the> total phospholipids, while t,he cellular fract’ion A showed lower values for phosphaticlyl choline and relatively higher values for phosphatidyl ethanolaminc and ephingomyelin (Table III). Thin layer chromatography of lipid clas.qc‘S rcvcaled tli:at, all fractions contained
NORMAL
AND
INACTIVATED
LUNG
SURF$CTANT
249
substantial amounts of triglycerides and cholesterol, which, however, have not been quantitatively evaluated. Measurement of total proteins revealed t’hat 74% of the noncellular proteins present in the bulk remained in the final supernatant C. The remaining 26% were
FIG. 1. Sediment B of minutes. The sediment is larly rounded bodies with fixed in 2% glutaraldehyde
lung washing obtained following centrifugation at 1OOOOOg for 90 mainly composed of whorl-like osmiophilic membranes and irreguthe characteristic features of the tubular myeloid figures. Sediment and postfixcd in 1% osmium tetroxide. ~12,100.
250
UALIY
1<1’ AL.
found in the sedimentary surfactant fraction I3 (Table II). However, rome contamination of the latter fraction with the protein-rich supcrnatant C was unavoidable. For this reason the sediment B was rcpeatcdly washed by rcsuqension in Ringer’s and centrifugation at 100,OOOg for 90 minutes. The protein
FIG. 2. Same sediment as Fig. 1. Depending on the plane of section the myeloid figures appear as periodic lamellae or a P square lattice. In addition, there are scattered dense particles, which are not due to staining nrt,ifact since tlrry could be readily seen in unstained preparations. Such pnrticlcs TVP~P often, but not consistently nsqociatcd with myeloid figures. X51.000.
NORMAL
FIG. 3. membranes x71,000.
AND
Sediment B of lung with a fingerprint-like
INACTIVATED
LUNG
washing showing arrangement,.
myeloid figures composed Sediment fixed in 1%
TABLE MINIMUM
SURFACE
TENSION
(7 MIN),
WASHING
(BULK)
of trilaminal unit osmium tetroxide.
II
PHOSPHOLIPID, AND
251
SURFACTANT
AND
PROTEIN
CENTRIFUCATION
VALUES
FOR NORM.\I.
LUNG
FRACTIONS
7 min per 2 mg ’ 5 mg dry dry weight weight __~ Bulk Sediment Sediment factant) Supernatant
A (cells) B (sed. surC
mg PLa (from ml bulk)
4.0
a Ww dry weight
mg Protein6 (from 40 ml bulk)
ProtZ/mg dry weight
-_
5-10 18 O-5
O-5 18 0
20.54 3.33 13.33
20
20
2.68
f 1.05c f 0.15d f.0.63 f
0.53
461 f 214 f 721 f
-
22 15 22
10.86 6.05 1.56
129 z& 31
4.42
f 0.20 + 0.23 zk 0.20
256 * 21 456 & 2 81 f 5
It
218 *
0.33
25
a PL = Phospholipids. * The values for sediment B are about 25y0 lower following resuspension in Ringer’s lactate solution and recentrifugation to eliminate contaminating proteins from supernatant C. c & indicates standard error. d The value 3.33 f 0.15 represents the phospholipid content of 99% of the cells present in 40 ml bulk. The remaining 1% of the cells (.04 mg PL) is recovered in the sediment B (13.33 f .63 mg PL) resulting in an insignificant contamination of the latter sediment with cellular phospholipids.
252
BALIS
ET AL.
TABLE PISRCENT Sphingomyelin BLllk Sediment Sediment Supernatant
1.8 9.5 1.0
A B C
OF TOTAL Phosphatidyl choline
III PHOSPHCJLIPIDS Phosphatidyl inositol
x1.4 52.4 90.2 100
2.7 3 fj 1.3 -
content of this washed sediment B was initially remained constant with additional washings. B. Thromboplastic
versus
surface
,activity
Phosphatidyl ethanolamine 5.0 14.0 2.4
Ca~a$~~~~fn_ identified PLj 8.3 20.3 4.4 -
reduced by about 25% and then
of lung extracts
Lung washing and cell-free sediment B of lung washing are both surface active with y min of 5-10 dynes/cm and O-5 dynes/cm, respectively, with 2 mg of lyophilized material. After mixing either of them with recalcified titrated plasma the clotting time was determined and it’ was found to be substantially shortened. Thus, the clotting time of recalcified titrated plasma previously diluted with equal volume of Ringer’s was 122 (27) seconds. By contrast, the clotting time was 27 (t-l) seconds and 16 (*I) seconds for the mixtures of plasma-washing, and plasma-sediment B, respectively. It is worth mentioning that lyophilization did not alter appreciably the thromboplast’ic activity of the above surface active extracts. On the other hand, after mixing recalcified titrated plasma with increasingly inactive fractions of lung washing, the clotting time was prolonged, proportionally to the elevation of minimum surface tension (Fig. 4). With the same st,andardized techniques the clotting time for the mixture of the recalcified citrat’ed plasma with brain thromboplastin (one stage prot,hrombin time of rabbit plasma) was 7.2 (kO.3) seconds. C. In vitro models Coagulative ~OTHLof surfactant inactivation. From t,he above findings it became evident that the rapid development of a clot in the mixture of surface act’ive extracts with recalcified titrated plasma was due to the strong thromboplastic activity of surfactant,. Further studies indicated that the surface properties of this mixture were drastically altered following clot formation. Thus, the y ,min of the eluate obtained from the misture after removal of the clot was 24-25 dynes/ cm. Moreover, ccntrifugation of the eluate yielded a sediment B which was inactive with y min of 20-25 dynes/cm. The alterations in the surface properties of the above mixtures before and aft’er clot formation could be actually observed in sequence in the surfactometer, by slowing down the process of coagulation. Thus, after mixing lung washing with hcparinized plasma in a ratio of 5:l the 7 min was initially about M-20 dynes/cm. However, within 30 minutes a clot developed in the washing-plasma mixture and the y min suddenly rose to 25 dynes/cm.
NORMAL
AND INACTIVATED
5
LUNG
SURFACTANT
15 20 25 10 MINlMUM SURFACE TENSION - DYNES/CM
253
30
FIO. 4. Clotting time following interaction of recalcified titrated plasma with normal lung washing (l), cell-free sediment B of lung washing (2), sediment A of lung washing (3), supernatant C of lung washing (4), and lung washing filtered with Seitz filter, 1 p pore (5).
Histologic examination revealed that the clots formed in the washing-plasma mixture contained fibrinous deposits and packed macrophages (Fig. 5). Actually, the macrophages in the washing-plasma mixture were almost completely incorporated in the clot, since centrifugation of the eluate following removal of the clot yielded sediments with practically no cells. By electron microscopy the above clots contained, in additon to fibrinous deposits and macrophages, numerous trapped myeloid figures, usually in the tubular form (Figs. 6 and 7). Identical changes but no cells were also observed in clots formed following interaction of plasma with cell-free sediment B. Lipid analysis revealed that 59% of phospholipid present in the washingplasma mixture was incorporated in the clot (Fig. 8). It should be noted that the presence of cells in the washing-plasma mixture was apparently not essential for the incorporation of surfactant phospholipids into the clot, since clots formed in mixtures of plasma with cell-free surfactant suspension contained at least 50% of the total phospholipids. Noncoagulative form of surfactant inactivation. After mixing lung washing with serum or titrated plasma in a ratio of 5: 1, the 7 min of the mixture was elevated (M-20 dynes/cm even with 20 mg of lyophilized material). By contrast,
254
BALM
ET $L.
FIG. 5. Section of clot formed following interaction of lung washing with plasma. The clot is composed of cells, granular material and fibrinous deposits. Epon embedded section (1 p thick) stained with toluidine blue. X460. FIG. 6. Section of the same block as Fig. 5. Portion of a macrophoge in association with extracellular myeloid figures, both “trapped” in the clot. The myeloid figures appear as periodic lamaellae or square lattice depending on the plane of section. X28,400.
NORMAL
AND
INACTIVATED
FI‘IO. 7. Same section as Fig. 5. Fibrinous X27.500.
LUNG
SURFACTANT
deposits in association
with
255
myeloid
figures.
256
BALIS
T
ET AL.
SEDIMENT B
FIG. 8. Distribution of phospholipids in fractions obtained from normal lung washing washing-serum mixture (II), and washing-plasma mixture (III).
(I),
sediment B of the mixture was strongly surface active with a 7 nzin of O-5 dynes/cm with 2 mg of lyophilized material. Fractions A and C were surface inactive with a y min greater than 20 dynes/cm. The results of lipid analysis showed that following differential sedimentation 54% of the surfactant phospholipids present, in the washing-serum mixture sedimented in the strongly surface active fraction B. However, 29% of the total phospholipids remained in the supernatant C as compared with 13% for fraction C of normal lung washing (Fig. 8). Further studies are in process to evaluate whether the above “excess” of nonsedimentable phospholipids in washing-serum mixture is related to altered sedimentation properties of the mixture or to other factors. Isotopic incorporation studies. The result’s of these studies confirmed the morphologic, surface tension, and biochemical data as to the distribution of surfactant phospholipids in the various fraction, q of normal and in vitro inactivated lung extracts. Thus, 62% of the total radioactivity was recovered in sediment B of washing-serum mixture as compared wit’h 72% for sediment B of normal lung washing (Fig. 9). By contrast, following interaction of plasma with either lung washing or cell-free surfactant suspension SO-SO%of the total radioactivity was incorporated in the clot. On the other hand, the eluate of the washing-plasma mixture yielded a sediment B which contained only 18% of the total radioactivity.
NORMAL
AND
INACTIVATED
LUNG
SURFACTANT
257
80
70
60
Fra. 9. Distribution of radioactivity from tritium labeled surfactant phospholipids recovered in sediment B following differential sedimentation of: (I) rabbit lung washing, (II) washing-serum mixture, and (III) eluate of washing-plasma mixture. In all above three systems the lung washing was obtained from rabbits killed 20 hours after intravenous injection of 3Hacetate.
DISCUSSION A. Qua&&ion
of nonnat lung surfactant
Lyophilized and nonlyophilized fractions of lung extracts have been previously utilized by several investigators for the isolation and study of surfactant lipoprotein (Bondurant and Miller, 1962; Finley et al., 1968; Said et al., 1968; Gladston et al., 1969). However, there are no published reports which meaningfully correlate at the quantitative level surface tension measurements with biochemical determinations of the surfactant components recovered from lung extracts. This correlation has been achieved in the present study which provides a structural, functional, and biochemical basis for the characterization and quantitation of surfactant lipoprotein isolated from lung extracts under normal and experimental conditions. The technique of differential sedimentation employed in this study was designed to isolate, characterize, and quantitate distinct constituents of the alveolar space. Thus, with the initial centrifugation of the bulk at 4509 for 5 minutes more than 99% of the cells were recovered in the surface inactive sediment A. No attempt was made to sediment all cellular elements of the bulk because pilot
258
BALIS
ET AL.
studies indicated that either longer centrifugation time or higher centrifugation speed resulted in lowering the minimum surface tension of the sediment A, indicating sedimentat’ion of surfactant. The findings that more than 80% of the noncellular phospholipids present in the bulk can be recovered in the surface active fraction B provides a unique opportunity to use t’his fraction for quantitative studies of surfactant changes. The lack of appreciable amounts of surfactant in fraction C makes this fraction especially valuable for the investigation of those noncellular constituents of the alveolar space which do not represent an integral part of the surfactant molecules. It is widely accepted that lung surfs&ant forms a layer at the air-tissue interface of the normal alveoli. There is also evidence that myeloid figures seen in alveolar spaces of lung tissues processed with standard electron microscopic techniques represent phospholipid components of the surfactant layer disrupted during tissue preparation (Kikkawa et al., 1968). Recent ultrastructural studies have utilized special fixat’ion procedures which preserve, at least in part, the alveolar air-liquid interface (Weibel and Gil, 1968; Kikkawa et al., 1970). With such procedures it has been possible to demonstrate an osmiophilic surface film at the air-liquid int’erface of normal alveoli (Weibel and Gil, 1968; Kikkawa et aE., 1970). Between this film and the alveolar epithelial lining there appears to exist an extracellular liquid layer, the so-called alveolar hypophase. Osmiophilic myeloid figures have been demonstrated in the hypophase (Weibel and Gil, 1968) which, therefore, may contain in suspension, surfactant presumably needed for the replenishment of the surface film during the respiratory cycle (Pattle, 1965). On the other hand, histochemical techniques at both light and electron microscopy levels have demonstrated the presence of a sialomucin-rich coat intimately associated with the plasma membrane of the alveolar epithelial cells (Macklin, 1954; Groniowski and Biczyskowa, 1964; Luke and Spicer, 1965; Groniowski and Biczyskowa, 1969; Kuhn, 1968; Bernstein et al., 1969). This mutinous coat is apparently not a unique feature of the alveolar epithelium since similar coating appears to cover the bronchial and intestinal epithelium as well as other cells (Pease, 1966). Special fixation procedures insuring preservation of the alveolar air-liquid interface are necessary for the demonstration of the surfactant layer but not for the mutinous coat. Moreover, the results of the present study suggest that removal of lung surfactant by alveolar lavage does not appreciably alter the distribution of the mutinous coat lining terminal air passages. These findings tend to suggest that the mutinous coat is an int’egral part of the epithelial plasma membranes rather than of the surfactant layer. This suggestion is further supported by t’he fact that a mutinous coat’ exists in the terminal air passages of immature fetal lungs which lack not only an air-liquid interface, but also appreciable amounts of surfactant (Bernstein et al., 1969). It should be mentioned, however, that there is evidence that the alveolar surfactant layer as well as lung extracts obtained by alveolar lavage contain mucopolysaccharides (Scarpelli, 1968; Bernstein et al., 1969; Kikkawa et al., 1970) which may conceivably originate from the mutinous coat. The hist,ochemical results of this study support the above concept, but they also emphasize the lack of firm basis for making the
NORMAL
AND INACTIVATED
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assumption that mucopolysaccharide changes of lung extracts necessarily reflect changes of the mutinous coat. In spite of the fact the myeloid figures have the structural features of phospholipids, there is still skepticism as to whether these structures indeed represent morphologic counterparts of surfactant phospholipids. Thus, it has been suggested that myeloid figures may actually originat,e from cellular debris in view of the observation that, while myeloid figures are sparse in normal alveoli, they arc seen in excessive amount in the alveolar exudate of lungs damaged by exposure to 100% oxygen (Harrison & Weibel, 1968). In addition, the myeloid figures in oxygen poisoned lungs often form cylindrical structures (tubular myeloid figures) which are uncommon in normal alveoli. In connection with these findings, it is of interest to mention t’hat earlier observations by Balis et al. (1966) indicated that collapsed alveoli of infant lungs with hyaline membrane disease consistently contain, during early stages of repair, a striking accumulation of packed myeloid figures in association with plasma proteins rather than cellular debris. Similar changes were observed in edematous dog lungs following unilateral pulmonary artery ligation (Balis et al., 1969)) and were thought to reflect a reactive, increased secretory activity of the type II epithelial cells. Recent studies by Kikkawa et al. (1968), have shown that the lecithin content in the tracheal lavage obtained from well expanded newborn rabbit lungs is greater than in the fluid-containing lungs of term rabbit fetus. However, the alveoli of the newborn lungs contain fewer myeloid figures than fetal alveoli. In addition, tubular mycloid figures are present in 2%day gestation rabbit fetus increasing rapidly toward term, but’ they are virtually absent in well expanded alveoli of normal newborn rabbits. Since the structure of liquid-crystalline phases of lipids, as judged by x-ray dcfraction data and electron microscopy (Luzzati and Husson, 1962; Stoeckenius, 1962), depends on various factors including the concentration of lipids in the water system, Kikkawa et al. (1968)) further suggested that the presence of fluid in the alveoli may result in a shift of the phase diagram of the pulmonary lipids. The above concept that the ultrastructural features of myeloid figures can be modified by factors which alter the phase of lipids in the water system is supported by the results of the present study which demonstrates that myeloid figures assume a tubular form in the cell-free, strongly surface active sediment B obtained from normal lung washings. It should be emphasized, that the liability of these structures with respect to lipid concentration, temperature of the system, presence of various cations, and other factors makes it exceedingly difficult to interpret electron microscopic observations relating to the myeloid figures (Luzzati and Husson, 1962; Stoeckenius, 1962). From all available data, therefore, it can be stated with certainty only that myeloid figures in the lung alveoli and in centrifugation sediments of lung washing represent phospholipid components of lung surfactant. With respect to the tubular myeloid figures recovered in the sediment B of our model, it can also be stated that ultracentrifugation probably affected the shape but not the organization of the structures, since similar tubular myeloid figures were found
260
BALE
ET AL.
to be incorporated into the clot following interaction washing with plasma. B. Thromboplastic activity
of noncentrifuged lung
of Iung surfactant
The findings of this study support the concept advanced by Taylor and Abrams (1966) that surfactant lipoprotein is a strongly thromboplastic substance which may, bherefore, rapidly result in intraalveolar fibrinous deposits in inflammatory and other conditions of the lung associated with significant transudation of plasma into the air spaces. Our results further indicate that thromboplastic activity of lung ext’ract’s as judged by substituting lyophilized or nonlyophilized lung extracts for brain thromboplastin in the one stage prothrombin time technique, increases in parallel with the surface activity of these extracts, being maximal, 16 (’ 1) seconds, in the strongly surface active sediment B. The above modified one stage prothrombin time technique, therefore, can be used as an additional parameter to be correlated with other criteria relating to surfactant properties. Since lung surfactant is a thromboplastic substance which may conceivably enter the circulation following lung trauma or disease, an invest,igation was recently undertaken to evaluate possible syst’emic effects of intravascular surfactant leakage. Preliminary results indicated that intravenous or left intracardial injection of surfactant lipoprotein results in pulmonary microthrombosis in association with endothelial damage, especially in animals pretreated with steroids (Rappaport et al., 1970). In view of the above findings it is worth further exploring the possibility that lung damage in various disease processes may predispose to the development of local and systemic thromboembolic phenomena. C. Reversible versus irreversible foms of swfactant inactivation It has been suggested that following interaction of fibrinogen with surfactant lipoprotein the latter is inactivated due to formation of a new substance (Taylor and Abrams, 1966). Our findings, however, indicate t’hat, the interaction of serum with surfactant results in a similar type of surfactant inactivation which should be considered as a reversible reaction since a surface active sediment B, rich in phospholipids, can be recovered from the above seemingly inactive mixture. By contrast, interact’ion of surfactant with plasma results in an irreversible form of surfactant inactivation due to clot formation. Our results further indicated t’hat the clot incorporates both surfactant phospholipids and cells, thus depleting the washing-plasma mixt’ure of its surfactant content and cellular elements. The above coagulative form of surfactant depletion may represent a mechanism of surfactant deficiency in lung diseases characterized by fibrinogen-rich alveolar exudate and formation of hyaline membranes (i.e., hyaline membrane disease of the newborn, primary atypical pneumonia, lung damage following oxygen toxicity, etc.). On the other hand, the seemingly reversible form of surfactant damage in the lung washing-serum mixture may occur in edematous conditions of the lung. This interpretation explains our previous observat’ion that inactive lung extracts from edematous dog lung following unilateral pulmonary artery ligation yield a strongly surface active sediment B (Balis et al., 1969). It is known that in lungs of infants with hyaline membrane disease (HMD),
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AND INACTIVATED
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alveolar macrophages are inconspicuous during the first 24 hours after delivery. There is also evidence that in addition to cell debris and plasma proteins (Balk et al., 1966) human hyaline membranes contain surfactant (Craig, 1964). On the basis of the present in vitro studies we propose that in HMD both surfactant and macrophages are incorporated in the intraalveolar fibrinous deposits which are eventually transformed into hyaline membranes following disintegration of the cellular elements. Further histochemical, ultrast’ructural, and biochemical studies are in progress to evaluate and compare the mechanism of membrane formation in in vitro models and in various human and experimental lung diseases. ACKNOWLEDGMENT The technical assistance of Elaine Smith, Maria Mezari, Binnie Lambert, is gratefully acknowledged. REFERENCES Surface phenomena
and Donna Janota
AVERY, M. E.. AND SAID, S. (1965). in lungs in health and disease. &Zeditine 44,503-526. BALIS, J. U., AND CONEN, P. E. (1964). The role of alveolar inclusion bodies in the developing lung. Lab. Invest. 13,1215-1229. BALIS, J. U., DELIVORIA, M., ANDCONEN, P. E. (1966). Maturation of postnatal human lung and the idiopathic respiratory distress syndrome. Lab. Invest. 15, 530-546. BALIS, J. U., AND &PPAPORT, E. S. (1969). In vitro inactivation of pulmonary surfactant and ‘(hyaline membrane” formation. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 26,745. BALIS, J. U., RAPPAPORT,E. S., PIFARRE, R., AND NEVILLE, W. E. (1969). Ultrastructural and surf&ant changes of the dog lung following pulmonary artery ligation. Lab. Invest. 20,574. BALIS, J. U., SHELLEY, S. A., MC&E, M. J., AND RAPPAPORT,E. S. (1970). Ultrastructure and quantitation of normal and in vitro inactivated lung surfactant. Amer. J. Pathol. 59, 95a. BENSCH. K., SCHAEFER, K., AND AVERY, M. E. (1964). Granular pneumonocytes: Electron microscopic evidence of their exocrinic function. Science 145,1318-1319. in the BERNSTEIN, J., YANG, S. S., HAHN, H. S., AND KIHECAWA, Y. (1969). Mucopolysaccharide pulmonary alveolus. I. Histochemical observations on the development of the alveolar lining layer. Lab. Invest. 21,420-425. BONDURANT,S., AND MILLER, D. A. (1962). A method for producing surface active extracts of mammalian lungs. J. Appl. Physiol. 17, 167-168. BUCKIXGHAM, S., HEINEMANN, H. O., SOMMERS,S. C., AND MCNARY, W. F. (1966). Phospholipid synthesis in the large pulmonary alveolar cell. Amer. J. Pathol. 48,1027-1041. CAVAGNA,G. A., VELASQUEZ, B. J., WETTON, R., AND DUBOIS, A. B. (1967). Cellular absorption of pulmonary surface-active material. J. Appl. Physiol. 22, 98%989. CLEMENTS, J. A., AND TIERNEY, D. F. (1965). Alveolar instability associated with altered surface tension. In “Handbook of Physiol.” (W. 0. Fenn and H. Rahn, eds.), Vol. 2, sec. 3. Amer. Physiol. Sot., Washington, D.C. CRAIG, J. (1964). The distribution of surface active material in the lungs of infants with and without respiratory distress. Biol. Neonatorum 7, 185-202. FINLEY, T. M., PRATT, S. A., LADMAN, A. J., BREWER, L., AND MCKAY, M. B. (1968). Morphological and lipid analysis of the alveolar lining material in dog lung. J. Lipid Res. 9, 357-365. FOLCH, J., LEES, M., AND SLOANE STANLEY, G. H. (1957). A sample method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-509. GLADSTON, M., SHAH, D. O., AND SHINOWARA. Y. (1969). Isolation and characterization of a lung lipoprotein surfactant. J. Colloid Interface Sci. 29,319-334. GIAMMONA, S. T., KERNER, D.. AND BONDURANT, S. (1965). Effect of oxygen breathing at atmospheric pressure on pulmonary surfactant. J. AppZ. Physiol. 20, 855-858. GREENFIELD, L. J., AND DUNCAN, R. C. (1968). Quantitative dynamic pulmonary surfactant. determinations with the pneumatic surfactometer. Surgery 64,351-358.
BALIS GRONIOWSKI,
lungs.
Nature
J.,
AND
204,
BICZYSKOWA,
W.
(1964).
ET
AL. Structure
of
the
alveolar
lining
film
of the
745-747.
J., AND BICZYSEOWA, D. (1969). The extraneous coat of lung alveoli. Lab. Zn20, 430-436. HARRISON, G. A., AXD WEIBEL, J. (1968). The membranous component of alveolar exudate. J. Ultrustruct. Res. 24, 334-342. HENRY, J. N. (1968). The effect of shocli on pulmonary alveolar surfactant. J. Trauma 8, 756-773. KIKKAWA, Y., MOTOI-AMA, E. K., AND COOK, C. D. (1965). The ultrastructure of the lungs of lambs. Amer. J. Pathol. 47,577~903. KIKKAWA, Y.. MOTOYARIA, E. K.. .~ND GLUCK, L. (1968). Study of the lungs of fetal and newborn rabbits: Morphological, biochemical and surface-physical development. Amer. J. Puthol. 52, 177-210. KIHKAWA, Y.. H.4~iv;. H. S.? YANG. S. S., AND BERNSTEIN, J. (1970). Mucopolysaccharide in the pulmonary alveolus. II. Electron microscopic ohsprvations. Lab. Znt’eat. 22, 272-280. Klaus. M., REISS, 0. K.. TOOLEY, W. H., PIEL, C., AND CLEDIENTS, J. A. (1962). Alveolar epithelial cell mitochondria as source of the surface-active lung lining. Science 137, 750-751. KUHN, C. III. (1968). Cytochemistry of pulmonary alveolar qlithelial cells. Amer. J. Pathol. 53,809~833. LOWRP, 0. H., ROSEBROUGH. N. J., FARR, A. L.. XXD RASDALL. K. J. (1951). Protein measurcment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. LUKE, J. I,.. AND SPICER. S. S. (1965). Histochemistry of surface epithelinl and pleural mucins in mammalian lung. Lab. Iwest. 14, 2101-2109. LUZZATI, V., AND HL-SSON. F. (1962). Thr structure of the liquid-crystalline phases of lipidwater systems. J. Cell Biol. 12, 207-219. M~CKLI~. C. C. (1954). The pulmonary alveolar mucoid film ant1 the pneumonocytes. Lancet 266, 1099-1104. MARTIN, J. B.. .~ND DOTI-, D. M. (1949). Determination of inorganic> 111losphate. And. Chem. 21,965-967. MARTIN. A. M.. Ja.. So~ow4r. H. B.. .~ND SI~IOKS, R. L. (1968). Pathologic anatomy of the lungs following shocB and trauma. J. Txzumn 8, 687-699. PATTLE. R. E. (1965). Surface lining of lung alveoli. Physiol. Rclr. 45, 4&79. PEASE, D. C. (1966). Polysaccharides associated with the exterior surface of epitheli:ll cells; kidney. intestine. brain. J. Ultmstruct. Res. 15,555-555. RAPPAPORT. E. S., MCCLX. M. J.. NOVAK, R., ASD B.41.1~. J. I-. (1070). Pulmonar)c1langc.Y following intravascular “leakage” of lung surfactant. Fed. Proc. Fed. Bmrr. Sot. Exp. Bid. 29, 489. SAID. 8. I.. HARLAN. W. R.. JR.. BURRE. G. W;.. .~ND %LIOTT. C. M. (1968). Surfacc tc~nsion. metabolic activity. and lipid composition of alveolar cells in washings from normal dog lungs and after pulmonary artery ligation. J. Clin. Invest. 47, 336-343. %‘,4RPEI,I.I, E. M. (1968). “The surfactanl s,vstem of the lung.” I,rn & Fcbiger, Philad~~lphi:r. SOHOKIS. S. P. (1966). A morphological and cytochemicnl stndF on the great alveolar ~~~11. .I. Hiptochem. Cytochem. 14, 884497. &Sl’ICER. 8s. S.. .4ND LILLIE. R. D. (1959). Saponification as n means of sc,lectivcly i-t>\-rr.qing 111~ meth>-lation bloclcade of tissue basophilin. J. Histochem. Cytochev~. 7, 123-125. SToErKExITTs. W. (1962). Some electron micro+ropical observations on licluid-crvst:lllinl, phas:Ps in lipid-water systems. J. Cc/l Biol. 12, 221-229. TAYLOR. F. R., JR., .4ND ABRA~VS. M. E. (1966). Effert of surface active 1ipol)rotein on clotting and fibrinolysis. and of fihrinogcn on surface tension of surface arti\-[s lipoprotein. Ampr. J. Med.40,346-350. WEIBEL. E. R,.. AXD GIL. J. (1968). Electron microzc*opic drmonstl:ltioll of an extra(ar11u1:lr duplex lining Ial-rr of alvco!i. Krsl). Physiol. 4, 42-57. WOLFE. W. G. .4~lD ISBISTOS. n. C.. JR. (1968). Tlrc rffrct of pulmon;lry rmbo1ism on pulmonarjsurfactani. Sl(Tgery 63, 312-319. WITTHIER. R. B. ( 1966). Two-dimmzional rl~rornatogra1)1~y on >i’ica gc1-]oadrd 1,;apcr fol the microanalysis of polar lipids. J. Lipid Rea. 7, 544-550. GROXIOWSKI,
vest.
AND
MOLECULAR
Mechanisms
of Damage
Ultrastructure
14,
PATHOLOGY
to the
and Quantitation inactivated
JOHN
Department
U.
BALIS,
o/Pathology, Maywood,
243-262
(1971)
Lung
Surfactant
of Normal
Loyola
of Chicago,
University
60153 und V.A. ZZeceivecl
and In Vifro
Lung Surfactant
SUE A. SHELLEY, MILES EDWARD S. RAPPAPORT
Illinois
System’
Hospital,
Decembe?
J.
MCCUE,
StTitch Bines,
AND
School
Illinois
of Medicine,
GOlgi
15,19iO
This study provides a structural, functional and biochemical basis for the characterization and quantitation of normal and in vitro inactivated lung surfactant. 1. Normal lung surfactant. Rabbit lung washing was centrifuged for 5 minutes at 4509 to obtain the cellular sediment A. The supernatant was centrifuged for 90 minutes at 100,OOOg yielding sediment B and supernatant C. All fractions were lyophilized and weighed. In addition, sediments A and B were processed for histochemistry and electron microscopy. Surface tension measurements revealed minimum surface tension (7 min) of O-5 dynes/cm with 5 mg of lung washing or with 2 mg of sediment B. By contrast, even with 20 mg of sediment A or supernatant C, y min was above 18 dynes/cm. By electron microscopy sediment B was composed mainly of myeloid figures. Biochemical determinations of lyophilized fractions revealed that more than 80% of the noncellular phospholipids present in the bulk sedimented into the strongly surface active sediment B, which was found to contain an average of 721 pg phospholipids/mg dry weight, with 90% of the phospholipid being phosphatidyl choline. Fractions A and C revealed relatively low phospholipid values, 214 pg/mg dry weight and 129 pg/mg dry weight, respectively. 2. In vitro inactivation of lung surfactant. (a) Interaction of lung washing with serum resulted in a seemingly reversible form of surfactant inactivation, since sediment B of the surface inactive washing-serum mixture revealed a high phospholipid content and a y min of O-5 dynes/cm with 2 mg of lyophilized material. (b) Following interaction of lung washing with heparinized or recalcified titrated plasma, a clot rapidly developed in the washing-plasma mixture due to the strong thromboplastic activity of surfactant. By electron microscopy the clot contained in addition to fibrinous deposits. packed macrophages, and numerous myeloid figures. Similar changes but no cells were observed in the clot formed in a mixture of cell-free surfactant suspension with plasma. Lipid analysis and isotopic tracing studies indicated that the clot incorporated 50-60% of the surfactant phopsholipids present in the plasma inactivated lung extracts. On the other hand the eluate of the washing-plasma mixture yielded a sediment B with low phospholipid content and y min of 20-25 dynes/cm. This form of surfactant inactivation can be defined, therefore, as a coagulative type of surfactant depletion, and may represent the mechanism of surfactant deficiency and membrane formation in hyaline membrane disease of the newborn and in other pathologic conditions of the lung associated with formation of hyaline membranes. 1 Study supported Health Service Grant
in part by HD-O4434-01.
USAMRDC
Contract 243
DADA
17-70-0041
and
U.S.
Public
244
lLU,TS E2’ AL.
Lung surfactant, is a complex? noncellular, surface active material which coats the alveoli of the normal lung (hvcry and Said, 1965; Pattle, 1965; Clements and Tierney, 1965; Scarpelli, 1968). The surface active component of the lung surfactant is a mixture of pl~ospholipitls:, hountl to proteins and carbollydrntes, and its main fuctioii is to reduce surface tension and stabilize air spaces. Hyalinc membrant~ discasc of the newborn is the most important clinicopathologic entity with an unclerlying surfactant defrct (Avery and Said, 1965). Lung surfactant is also d:maged in shock (Henry! 1968; Mart,in et nl., 1968)) pulmonary embolism (Greenfield and Duncan, 1968; Wolfe and Sabiston, 1968)) oxygen toxicity (Giammona et rrl., 1965) and in other pat,hologic conditions of the lung characterized by atrlccta. *‘u, 1, edema, h(~morrhages,and often formation of hyaline membranes in alveolar spaces. The pathogenctic interrelationships bct’ween the above lung changes and surfactant deficiency remains uncertain, in spite of extensive work in this field. From various lines of evidence it’ appears that the lung surfactant is synt’hesizcd and aecrctcd by the type II epithelial cells (Macklin, 1954; Klaus et ul., 1962; Buckingham et nl., 1966; Balis and Concn, 1964; Bensch et (11.,1964; Kikkawa et (11.,1965; Sorokin, 1966; Kikkawa et al., 1968; Kuhn, 1968)) but primary surfactant deficiency due to impaired synthesis of surfactant has never been convincingly demonstrated. On the other hand, recent studies appear to support the concept that interaction of t,he lung surfact’ant with blood constituents may lead to damage or inactivation of lung surfactant (Tnylor and Ahrams, 1966). T_;nfort~uuatcly, at, present. it is not’ possible to clefinc and differentiate distinct forms of aurfnctant, tlamngc or inactivation. This is due to the fact that the structural fcaturcs of normal and altered surfnctant hare not been correlated wit,11quantitative, functional, and biochemical parameters relating to surfactant activity or deficiency. From studies in our laboratory, methods have been clrvclol~tl which provide the opportunity to (a ) identify, recover, and quantitate even minute amounts of surface active fractions from lung extracts! and (b) meaningfully correlate morl)hologic charnctt%tics of hurfac;:\nt’ components with quantitative surface tension measurements and biochemical data. Furt,hcrmore, using the above parnmctcrs and an i,,. zbitro model (Balis ant1 Rappaport, 1969; Balis et (II., 1970), it has been possible to study and characterize reversible and irreversible forms of surfactant inactivation following intcraction of blood constituents with lung surfactant.
Preparation of lung washing and its centrifugation fractions. Thirty-five malt albino rabbits weighing I .4-1.6 kg were ancAlctizcd by intravenous injection of sodium pentobarbital. Thr animals wcrc cx.sanguinated by cutting the aorta nncl inferior vena cara, and before opening the chest, cavity, the trachea was clwm1~~l to prevent the entry of blood into the tracheobronchial tree. Following removal of the lungs, Ringer’s lactate solution (25 ml/kg body weight) was introducccl into the lungs through a cannula in the trachea. The buffer solution was instilled over a period of 1 minute, was allowl-cclto remain in the lungs for 30 seconds, ant1 wa8 removed by gravity drainage and gcnt’le pressure. This procedure was repeated and from a total of about 70 ml of buffer solution introduced into the
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AND INACTIVATED
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lungs 48-50 ml of white, opalescent washing was recovered. Ringer’s lactate rather than isotonic saline was used for the alveolar lavage because pilot studies indicated that saline results in significant damage of the alveolar macrophages. The washing was filtered through a four-layered gauze to remove mucus, and it is designated herein as bulk. A 5-ml sample of the bulk was placed separately and the remainder was centrifuged at 450g for 5 minutes to obtain the cellular sediment A. A cell count of the bulk and the supernatant obtained following the above initial centrifugation revealed an average of 50,000 and 200 cells per mm3, respectively. Thus, more than 99% of the cells present in the bulk sedimented into the fraction A. The cell-depleted supernatant was further centrifuged at 100,OOOgfor 90 minutes at 4°C to obtain the sediment B and the final supernatant C. In some experiments, the bulk was initially centrifuged at 50009, 4°C for 60 minutes prior to ultracentrifugation in order to obtain a cell-free sediment B. Samples of 5 ml from the bulk and the supernatant C were lyophilized. The sediments A and B were also lyophilized following resuspension in 5 ml Ringer’s lactate. All above fractions were placed prior to lyophilization in dehumidified and preweighed 10 ml bottles. The dry weight of the lyophilized fraction was carefully determined by subtracting the initial weight of the bottles from their final weight. Since all fractions were suspended in Ringer’s lactate before lyophilization, the dry weight of their buffer content was determined and subtracted from the final dry weight of the lyophilized fractions. This determination was possible in view of the fact that (a) the dry weight of Ringer’s lactate was found to be constant, 9.8 + .Ol mg/ml, and (b) the concentration of sodium in all Ringer’s suspendedfractions was identical (136 f 2 mEq/liter) . Thromboplastic activity of lung extracts. Citrated rabbit plasma was mixed with the following lung extracts, the minimum surface tension (v nzin) of which was previously determined using 2-3 mg of lyophilized materials: (1) bulk (y ,min = 5-10 dynes/cm), (2) cell-free sediment B (7 min = O-5 dynes/cm), (3) supernatant C (7 min = 20-21 dynes/cm), (4) cellular sediment A (y min = 18-19 dynes/cm), and (5) lung washing filtered with Seitz filters having 1~ pores (y min = 22-24 dynes/cm). The mixtures of the above extracts with plasma were tested for surface activity. In addition, the clotting time of the mixtures of recalcified titrated plasma with extracts l-5 was determined under standardized conditions using a fibrometer precision coagulation timer. The clotting time of these mixtures was compared with the conventional Quick one stage prothrombin time of rabbit plasma in order to evaluate the thromboplastic activity of each of the above lung extracts. In vitro models. In one set of experiments, rabbit lung washing was thoroughly mixed with serum or titrated plasma in a ratio of 5:1, and from these mixtures fractions A, B, and C were obtained using the technique of differential sedimentation. Cell counts revealed that sediment A contained more than 99% of the cells present in the washing-serum mixture. In a second series of experiments, lung washing or resuspended cell-free sediment B of lung washing was mixed with heparinized plasma in a ratio of 5:l. Mixtures of the above lung extracts with titrated, recalcified plasma were also used. Following clot formation, the eluate was fractionated as the washing-serum mixture.
246
BrZI,IS
B2’
AI,.
The technique of differential sedimentation used in the in zlitro models was the same as that employed for the normal lung washing, with the exception that sediment B was repeatedly resuspended in Ringer’s lactate solution and ccnt,rifuged in order to eliminate cont’aminating serum constituents. In some cxperimerits, the pl~os1~l~olil~ids of lung cxtrants were previously labeled by intravenous injection of rabbits, 20 hours prior to sacrifice, with tritiated sodium acetate (specific activity of 2.66 Ci/mnl), 250 &i/kg body weight. These isotopic studies were performed to evaluate and compare the distribution of radioactively labeled phospholipids in (a) the ccntrifugation fractions of normal lung washing and washing-serum mixture and (b) the clot and centrifugat,ion fractions of the eluate obtained following interaction of plasma with surface active lung extracts. Electron ?nicroscopy. Fragments of clot’ and nonlyophilizcd sediments A and B, obtained respectively by centrifugation of bulk at 450g for 5 minutes and 100,OOOgfor 90 minutes, were fixed in 1% buffered osmium tetroxicle with and without prior fixation in 256 buffered glut~araldehydc. Following dehydration in ascending concentrations of ethanol solution, the sediments were embeddctl in Epon 812. Ultrathin sections were examined with an RC;2 EMU-SF electron microscope after staining wit’h uranyl acetate and lead cit’rate. In addition, 1-p thick sections were stained with toluidine blue for light microscopy. Histochemist~y. Sediments A and B as well as lung samples obt’ained before and after lavage were fixed in 10% buffered formalin with 2% calcium acetate. Paraffin embedded sections were stained with the periodic-acid Schiff reaction (PAS), diastasc-P’ad, and Halt colloidal iron as modified by Xowry. To idcntify sialomucins sections were digested with I’ibrio cholerne sialidase (General Biochemicals, Inc., Chagrin Falls, Ohio) as described by Luke and Spicer (1965). In some sections blocking by methylation was pcrformcd and the sections were stained with Mowry’s colloidal iron before and after saponification according to the method of Spicer and Lillic (1959). Resuspended sediments A and B were dried on slides, and following formalin fixation and dehydration were stained using the same histochemical procedure described abore for paraffin sections. In addition, dried sediments were treated following fixation and dehydration with chloroforni-methanol (2: 1) for 16 hours to extract the lipids, and stained with PAS and Mowry’s colloidal iron. Test for surfactant function. A Greenfield pneumatic surfactometer was used for the surface tension studies. Liquid bulk was cxamincd as described by Grccsnfield and Duncan (1968). Lyophilized materials were spread on the surface of Ringer’s solution in the trough, and for each fraction the minimum surfarc tension (y ~min) was determined with 2-20 mg of lyophilized material. Each sample was allowed to cycle repeatedly at’ 3 minute cycle times until it’ showed n stable replication of the hysteresis loop. Biochemical annly~sis. Tot,al lipid s were cstractod from the, lyopliilized fractions with chloroform-met~hanol by the method of Folch et rrl. (1957). The extracted lipids were dissolved in a known volume of chloroform-methanol and measured aliquots were used for separation of lipid classes, separation of t,he phospholipids, and total phosphorus determinations. Lipid classeswere scparatccl on silica gel G plates (Analtech, Inc.). The Polvent was composed of petroleum
NORMAL
AND
INACTIVATED
LUNG
247
SURFACTANT
ether, ether, and acetic acid (80:20:1) and the plates were developed for 7 to 8 cm. The lipids were visualized by spraying with 10% phosphomolybdic acid in ethanol followed by heating at 80°C for 5 minutes. Phospholipids were separated by two-dimensional chromatography on Whatman SG-81 silica gel-loaded paper as described by Wuthier (1966). Development in the first direction was with a solvent composed of chloroform, methanol, diisobutylketone, acetic acid, and water (90:30:60:40:8). After drying, the paper was developed in the second direction with chloroform, methanol, diisobutylketone, pyridine, and .5 M, pH 10.4, ammonium chloride buffer (60: 35: 50: 70: 12). For phosphorus determinations of the phospholipid spots, the dried papers were stained with Rhodamine 6G and examined under UV light. Total lipid phosphorus and phosphorus in each of the separated phospholipids were measured by a modification of the method of Martin and Doty (1949) as described by Wuthier (1966). The phospholipid content was calculated assuming that phospholipids contained 4% phosphorus. Total protein was measured by the method of Lowry et al. (1951). Radioactivity was measured using a Tri-Carb scintillation counter. RESULTS A. Normal
lung surfactant
Histochemical and electron microscopic findings. By light microscopy the surfact active sediment B appeared as a granular material intermixed with occasional cells. The granular material was positive (red) with PAS, and blue with the Mowry’s colloidal iron stain. The Mowry’s staining reaction could be blocked by methylation, but it was restored with saponification. Sialidase digestion also blocked the staining but pretreatment of slides with chloroform-methanol had no effect on either PAS or Mowry’s colloidal iron (Table I). In the surface inactive cellular sediment A, no extracellular elements wit,h the above histochemical characteristics could be detected. In lung tissues obtained before and after alveolar lavage, staining of sections with Mowry’s colloidal iron failed to demonstrate any alteration of the mutinous coat which is know to cover the air passages (see Discussion). By electron microscopy, the granular material seen in sediment B was found to be composed of osmiophilic myeloid figures, in association with some moderately TABLE ST.ZINING
RKACTIONS
I
OF SEDIMKNTS
FROM
LUNG
WASHING
Mowry’s ‘As
Sediment A 1. Cells 2. Extracellular spaces Sediment, B (surfact,ant)
Mowry’s
2iy;
+ -
+ -
+ -
+
+
-
Methylation
-
PAS ChloroformMethan01
tion
ChloroformMeth-
+ -
+ -
+ -
+
+
+
‘:?a:-
an01
248
BSJdS ET AL.
dense amorphous material, and occasional fragments of cells. Thcbmyeloid figures appeared as either m-horl-like membranous profiles or as irregularly shaped granules or globular bodies. The latter structures rcrcaled a characteristic orderly pattern in forms of periodic lamellac or square lattice, depending on the plane of ,qcction (Figs. l-3). Cell-free scdimcnt B revealed similar ultra~tructural fcaturca with the exception that ccl1 fragmcntjs were virt’ually absent. Swf.nce tension. ttlecrsuwttlents. Previous stutlics have demoiist~rateclthat surfactant readily sediments with ccutrifugation (Finley et crl., 1968; Cavagna et al., 1967; Galdston et al., 1969; Said et al., 19681. The results of the present8 work, however, indicate that with the initial ccntrifugation of the bulk at 4509 for 5 minutes, very little surfactant is lost in t,he cellular sediment A since the minimum surface tension (7 tnin) of this fraction is clevatcd (above 18 dynes/ cm) with 2, 5, and cvcn 20 mg of lyophilized material (Table II). Similarly, the final supcrnatant C is surface inactive with 7 ,?zin above 20 dynes/cm. By contrast, lyophilized material from sediment B obtained following ccntrifugat’ion at 100,OOOgfor 90 minutes was strongly surface active, lowering surface tension below 5’ dynes/cm wit#h 2 mg of lyophilized material containing an average of 500 pg of surface active material. Thcsc findings suggcbstthat most of the surfactant present in the bulk can bc rccorc~recl in the s:cdiment B. It al~oulcl be mcntionccl that the presence of some cells in sediment B was insignificant in terms of surface tension, since following complctc removal of t’hese cells there was no tletectablc increase in the surface activity of the xbovc scdimcnt. Further studies revealed that while the surfarc act)ivity of the bulk diluted in various proportions with Ringer’ s was rcclucecl proportionally to the degree of dilution, the sediment B obtained from the above diluted surfactant suspension was strongly surface act)ive, lowering surface tension below 5 dynes/cm with 2 mg of lyophilized material. This suggests that’ the surface act’ivity of sediment B is constant’ on a dry w(>ight#basis provided that adequate quant,itics of sedimentable surfactant are present in the initial bulk. Biochemical determinations. The above findings correlated well with the pho~pholipid determinations, which yielded consistent and readily reproducible range of values for the bulk and its ccntrifugation fractions (Table II). The rrsult:: indicated that with the technique of cliffcrent,ial sedimentation employed herein, 17% and 70% of the phospholipids present, in the bulk wcrc setliment8edinto the fractions A and B, respectively. Since the cellular fraction iz contains relatively little surfactant, as judged by the surface tension measurements and the elec,tron microscopic findings (see below), it is reasonable to assumethat the phospholipid content of this fraction only reflect s its cellular origin. From the remaining phospholipids of the bulk, therefore, more than 80% was rccovcrccl in the surface active, cell depleted sediment B. The latter fraction also revealed the highest values of phospholipids per milligram of dry weight, and these values did not increase further when cell-free sediment B was used. The results of two-climcxsional chromatography indicated that in either cell-fret or cell clepletccl scdimcnt B, phosphaticlyl choline represented 90% of the> total phospholipids, while t,he cellular fract’ion A showed lower values for phosphaticlyl choline and relatively higher values for phosphatidyl ethanolaminc and ephingomyelin (Table III). Thin layer chromatography of lipid clas.qc‘S rcvcaled tli:at, all fractions contained
NORMAL
AND
INACTIVATED
LUNG
SURF$CTANT
249
substantial amounts of triglycerides and cholesterol, which, however, have not been quantitatively evaluated. Measurement of total proteins revealed t’hat 74% of the noncellular proteins present in the bulk remained in the final supernatant C. The remaining 26% were
FIG. 1. Sediment B of minutes. The sediment is larly rounded bodies with fixed in 2% glutaraldehyde
lung washing obtained following centrifugation at 1OOOOOg for 90 mainly composed of whorl-like osmiophilic membranes and irreguthe characteristic features of the tubular myeloid figures. Sediment and postfixcd in 1% osmium tetroxide. ~12,100.
250
UALIY
1<1’ AL.
found in the sedimentary surfactant fraction I3 (Table II). However, rome contamination of the latter fraction with the protein-rich supcrnatant C was unavoidable. For this reason the sediment B was rcpeatcdly washed by rcsuqension in Ringer’s and centrifugation at 100,OOOg for 90 minutes. The protein
FIG. 2. Same sediment as Fig. 1. Depending on the plane of section the myeloid figures appear as periodic lamellae or a P square lattice. In addition, there are scattered dense particles, which are not due to staining nrt,ifact since tlrry could be readily seen in unstained preparations. Such pnrticlcs TVP~P often, but not consistently nsqociatcd with myeloid figures. X51.000.
NORMAL
FIG. 3. membranes x71,000.
AND
Sediment B of lung with a fingerprint-like
INACTIVATED
LUNG
washing showing arrangement,.
myeloid figures composed Sediment fixed in 1%
TABLE MINIMUM
SURFACE
TENSION
(7 MIN),
WASHING
(BULK)
of trilaminal unit osmium tetroxide.
II
PHOSPHOLIPID, AND
251
SURFACTANT
AND
PROTEIN
CENTRIFUCATION
VALUES
FOR NORM.\I.
LUNG
FRACTIONS
7 min per 2 mg ’ 5 mg dry dry weight weight __~ Bulk Sediment Sediment factant) Supernatant
A (cells) B (sed. surC
mg PLa (from ml bulk)
4.0
a Ww dry weight
mg Protein6 (from 40 ml bulk)
ProtZ/mg dry weight
-_
5-10 18 O-5
O-5 18 0
20.54 3.33 13.33
20
20
2.68
f 1.05c f 0.15d f.0.63 f
0.53
461 f 214 f 721 f
-
22 15 22
10.86 6.05 1.56
129 z& 31
4.42
f 0.20 + 0.23 zk 0.20
256 * 21 456 & 2 81 f 5
It
218 *
0.33
25
a PL = Phospholipids. * The values for sediment B are about 25y0 lower following resuspension in Ringer’s lactate solution and recentrifugation to eliminate contaminating proteins from supernatant C. c & indicates standard error. d The value 3.33 f 0.15 represents the phospholipid content of 99% of the cells present in 40 ml bulk. The remaining 1% of the cells (.04 mg PL) is recovered in the sediment B (13.33 f .63 mg PL) resulting in an insignificant contamination of the latter sediment with cellular phospholipids.
252
BALIS
ET AL.
TABLE PISRCENT Sphingomyelin BLllk Sediment Sediment Supernatant
1.8 9.5 1.0
A B C
OF TOTAL Phosphatidyl choline
III PHOSPHCJLIPIDS Phosphatidyl inositol
x1.4 52.4 90.2 100
2.7 3 fj 1.3 -
content of this washed sediment B was initially remained constant with additional washings. B. Thromboplastic
versus
surface
,activity
Phosphatidyl ethanolamine 5.0 14.0 2.4
Ca~a$~~~~fn_ identified PLj 8.3 20.3 4.4 -
reduced by about 25% and then
of lung extracts
Lung washing and cell-free sediment B of lung washing are both surface active with y min of 5-10 dynes/cm and O-5 dynes/cm, respectively, with 2 mg of lyophilized material. After mixing either of them with recalcified titrated plasma the clotting time was determined and it’ was found to be substantially shortened. Thus, the clotting time of recalcified titrated plasma previously diluted with equal volume of Ringer’s was 122 (27) seconds. By contrast, the clotting time was 27 (t-l) seconds and 16 (*I) seconds for the mixtures of plasma-washing, and plasma-sediment B, respectively. It is worth mentioning that lyophilization did not alter appreciably the thromboplast’ic activity of the above surface active extracts. On the other hand, after mixing recalcified titrated plasma with increasingly inactive fractions of lung washing, the clotting time was prolonged, proportionally to the elevation of minimum surface tension (Fig. 4). With the same st,andardized techniques the clotting time for the mixture of the recalcified citrat’ed plasma with brain thromboplastin (one stage prot,hrombin time of rabbit plasma) was 7.2 (kO.3) seconds. C. In vitro models Coagulative ~OTHLof surfactant inactivation. From t,he above findings it became evident that the rapid development of a clot in the mixture of surface act’ive extracts with recalcified titrated plasma was due to the strong thromboplastic activity of surfactant,. Further studies indicated that the surface properties of this mixture were drastically altered following clot formation. Thus, the y ,min of the eluate obtained from the misture after removal of the clot was 24-25 dynes/ cm. Moreover, ccntrifugation of the eluate yielded a sediment B which was inactive with y min of 20-25 dynes/cm. The alterations in the surface properties of the above mixtures before and aft’er clot formation could be actually observed in sequence in the surfactometer, by slowing down the process of coagulation. Thus, after mixing lung washing with hcparinized plasma in a ratio of 5:l the 7 min was initially about M-20 dynes/cm. However, within 30 minutes a clot developed in the washing-plasma mixture and the y min suddenly rose to 25 dynes/cm.
NORMAL
AND INACTIVATED
5
LUNG
SURFACTANT
15 20 25 10 MINlMUM SURFACE TENSION - DYNES/CM
253
30
FIO. 4. Clotting time following interaction of recalcified titrated plasma with normal lung washing (l), cell-free sediment B of lung washing (2), sediment A of lung washing (3), supernatant C of lung washing (4), and lung washing filtered with Seitz filter, 1 p pore (5).
Histologic examination revealed that the clots formed in the washing-plasma mixture contained fibrinous deposits and packed macrophages (Fig. 5). Actually, the macrophages in the washing-plasma mixture were almost completely incorporated in the clot, since centrifugation of the eluate following removal of the clot yielded sediments with practically no cells. By electron microscopy the above clots contained, in additon to fibrinous deposits and macrophages, numerous trapped myeloid figures, usually in the tubular form (Figs. 6 and 7). Identical changes but no cells were also observed in clots formed following interaction of plasma with cell-free sediment B. Lipid analysis revealed that 59% of phospholipid present in the washingplasma mixture was incorporated in the clot (Fig. 8). It should be noted that the presence of cells in the washing-plasma mixture was apparently not essential for the incorporation of surfactant phospholipids into the clot, since clots formed in mixtures of plasma with cell-free surfactant suspension contained at least 50% of the total phospholipids. Noncoagulative form of surfactant inactivation. After mixing lung washing with serum or titrated plasma in a ratio of 5: 1, the 7 min of the mixture was elevated (M-20 dynes/cm even with 20 mg of lyophilized material). By contrast,
254
BALM
ET $L.
FIG. 5. Section of clot formed following interaction of lung washing with plasma. The clot is composed of cells, granular material and fibrinous deposits. Epon embedded section (1 p thick) stained with toluidine blue. X460. FIG. 6. Section of the same block as Fig. 5. Portion of a macrophoge in association with extracellular myeloid figures, both “trapped” in the clot. The myeloid figures appear as periodic lamaellae or square lattice depending on the plane of section. X28,400.
NORMAL
AND
INACTIVATED
FI‘IO. 7. Same section as Fig. 5. Fibrinous X27.500.
LUNG
SURFACTANT
deposits in association
with
255
myeloid
figures.
256
BALIS
T
ET AL.
SEDIMENT B
FIG. 8. Distribution of phospholipids in fractions obtained from normal lung washing washing-serum mixture (II), and washing-plasma mixture (III).
(I),
sediment B of the mixture was strongly surface active with a 7 nzin of O-5 dynes/cm with 2 mg of lyophilized material. Fractions A and C were surface inactive with a y min greater than 20 dynes/cm. The results of lipid analysis showed that following differential sedimentation 54% of the surfactant phospholipids present, in the washing-serum mixture sedimented in the strongly surface active fraction B. However, 29% of the total phospholipids remained in the supernatant C as compared with 13% for fraction C of normal lung washing (Fig. 8). Further studies are in process to evaluate whether the above “excess” of nonsedimentable phospholipids in washing-serum mixture is related to altered sedimentation properties of the mixture or to other factors. Isotopic incorporation studies. The result’s of these studies confirmed the morphologic, surface tension, and biochemical data as to the distribution of surfactant phospholipids in the various fraction, q of normal and in vitro inactivated lung extracts. Thus, 62% of the total radioactivity was recovered in sediment B of washing-serum mixture as compared wit’h 72% for sediment B of normal lung washing (Fig. 9). By contrast, following interaction of plasma with either lung washing or cell-free surfactant suspension SO-SO%of the total radioactivity was incorporated in the clot. On the other hand, the eluate of the washing-plasma mixture yielded a sediment B which contained only 18% of the total radioactivity.
NORMAL
AND
INACTIVATED
LUNG
SURFACTANT
257
80
70
60
Fra. 9. Distribution of radioactivity from tritium labeled surfactant phospholipids recovered in sediment B following differential sedimentation of: (I) rabbit lung washing, (II) washing-serum mixture, and (III) eluate of washing-plasma mixture. In all above three systems the lung washing was obtained from rabbits killed 20 hours after intravenous injection of 3Hacetate.
DISCUSSION A. Qua&&ion
of nonnat lung surfactant
Lyophilized and nonlyophilized fractions of lung extracts have been previously utilized by several investigators for the isolation and study of surfactant lipoprotein (Bondurant and Miller, 1962; Finley et al., 1968; Said et al., 1968; Gladston et al., 1969). However, there are no published reports which meaningfully correlate at the quantitative level surface tension measurements with biochemical determinations of the surfactant components recovered from lung extracts. This correlation has been achieved in the present study which provides a structural, functional, and biochemical basis for the characterization and quantitation of surfactant lipoprotein isolated from lung extracts under normal and experimental conditions. The technique of differential sedimentation employed in this study was designed to isolate, characterize, and quantitate distinct constituents of the alveolar space. Thus, with the initial centrifugation of the bulk at 4509 for 5 minutes more than 99% of the cells were recovered in the surface inactive sediment A. No attempt was made to sediment all cellular elements of the bulk because pilot
258
BALIS
ET AL.
studies indicated that either longer centrifugation time or higher centrifugation speed resulted in lowering the minimum surface tension of the sediment A, indicating sedimentat’ion of surfactant. The findings that more than 80% of the noncellular phospholipids present in the bulk can be recovered in the surface active fraction B provides a unique opportunity to use t’his fraction for quantitative studies of surfactant changes. The lack of appreciable amounts of surfactant in fraction C makes this fraction especially valuable for the investigation of those noncellular constituents of the alveolar space which do not represent an integral part of the surfactant molecules. It is widely accepted that lung surfs&ant forms a layer at the air-tissue interface of the normal alveoli. There is also evidence that myeloid figures seen in alveolar spaces of lung tissues processed with standard electron microscopic techniques represent phospholipid components of the surfactant layer disrupted during tissue preparation (Kikkawa et al., 1968). Recent ultrastructural studies have utilized special fixat’ion procedures which preserve, at least in part, the alveolar air-liquid interface (Weibel and Gil, 1968; Kikkawa et al., 1970). With such procedures it has been possible to demonstrate an osmiophilic surface film at the air-liquid int’erface of normal alveoli (Weibel and Gil, 1968; Kikkawa et aE., 1970). Between this film and the alveolar epithelial lining there appears to exist an extracellular liquid layer, the so-called alveolar hypophase. Osmiophilic myeloid figures have been demonstrated in the hypophase (Weibel and Gil, 1968) which, therefore, may contain in suspension, surfactant presumably needed for the replenishment of the surface film during the respiratory cycle (Pattle, 1965). On the other hand, histochemical techniques at both light and electron microscopy levels have demonstrated the presence of a sialomucin-rich coat intimately associated with the plasma membrane of the alveolar epithelial cells (Macklin, 1954; Groniowski and Biczyskowa, 1964; Luke and Spicer, 1965; Groniowski and Biczyskowa, 1969; Kuhn, 1968; Bernstein et al., 1969). This mutinous coat is apparently not a unique feature of the alveolar epithelium since similar coating appears to cover the bronchial and intestinal epithelium as well as other cells (Pease, 1966). Special fixation procedures insuring preservation of the alveolar air-liquid interface are necessary for the demonstration of the surfactant layer but not for the mutinous coat. Moreover, the results of the present study suggest that removal of lung surfactant by alveolar lavage does not appreciably alter the distribution of the mutinous coat lining terminal air passages. These findings tend to suggest that the mutinous coat is an int’egral part of the epithelial plasma membranes rather than of the surfactant layer. This suggestion is further supported by t’he fact that a mutinous coat’ exists in the terminal air passages of immature fetal lungs which lack not only an air-liquid interface, but also appreciable amounts of surfactant (Bernstein et al., 1969). It should be mentioned, however, that there is evidence that the alveolar surfactant layer as well as lung extracts obtained by alveolar lavage contain mucopolysaccharides (Scarpelli, 1968; Bernstein et al., 1969; Kikkawa et al., 1970) which may conceivably originate from the mutinous coat. The hist,ochemical results of this study support the above concept, but they also emphasize the lack of firm basis for making the
NORMAL
AND INACTIVATED
LUNG
SURFACTANT
259
assumption that mucopolysaccharide changes of lung extracts necessarily reflect changes of the mutinous coat. In spite of the fact the myeloid figures have the structural features of phospholipids, there is still skepticism as to whether these structures indeed represent morphologic counterparts of surfactant phospholipids. Thus, it has been suggested that myeloid figures may actually originat,e from cellular debris in view of the observation that, while myeloid figures are sparse in normal alveoli, they arc seen in excessive amount in the alveolar exudate of lungs damaged by exposure to 100% oxygen (Harrison & Weibel, 1968). In addition, the myeloid figures in oxygen poisoned lungs often form cylindrical structures (tubular myeloid figures) which are uncommon in normal alveoli. In connection with these findings, it is of interest to mention t’hat earlier observations by Balis et al. (1966) indicated that collapsed alveoli of infant lungs with hyaline membrane disease consistently contain, during early stages of repair, a striking accumulation of packed myeloid figures in association with plasma proteins rather than cellular debris. Similar changes were observed in edematous dog lungs following unilateral pulmonary artery ligation (Balis et al., 1969)) and were thought to reflect a reactive, increased secretory activity of the type II epithelial cells. Recent studies by Kikkawa et al. (1968), have shown that the lecithin content in the tracheal lavage obtained from well expanded newborn rabbit lungs is greater than in the fluid-containing lungs of term rabbit fetus. However, the alveoli of the newborn lungs contain fewer myeloid figures than fetal alveoli. In addition, tubular mycloid figures are present in 2%day gestation rabbit fetus increasing rapidly toward term, but’ they are virtually absent in well expanded alveoli of normal newborn rabbits. Since the structure of liquid-crystalline phases of lipids, as judged by x-ray dcfraction data and electron microscopy (Luzzati and Husson, 1962; Stoeckenius, 1962), depends on various factors including the concentration of lipids in the water system, Kikkawa et al. (1968)) further suggested that the presence of fluid in the alveoli may result in a shift of the phase diagram of the pulmonary lipids. The above concept that the ultrastructural features of myeloid figures can be modified by factors which alter the phase of lipids in the water system is supported by the results of the present study which demonstrates that myeloid figures assume a tubular form in the cell-free, strongly surface active sediment B obtained from normal lung washings. It should be emphasized, that the liability of these structures with respect to lipid concentration, temperature of the system, presence of various cations, and other factors makes it exceedingly difficult to interpret electron microscopic observations relating to the myeloid figures (Luzzati and Husson, 1962; Stoeckenius, 1962). From all available data, therefore, it can be stated with certainty only that myeloid figures in the lung alveoli and in centrifugation sediments of lung washing represent phospholipid components of lung surfactant. With respect to the tubular myeloid figures recovered in the sediment B of our model, it can also be stated that ultracentrifugation probably affected the shape but not the organization of the structures, since similar tubular myeloid figures were found
260
BALE
ET AL.
to be incorporated into the clot following interaction washing with plasma. B. Thromboplastic activity
of noncentrifuged lung
of Iung surfactant
The findings of this study support the concept advanced by Taylor and Abrams (1966) that surfactant lipoprotein is a strongly thromboplastic substance which may, bherefore, rapidly result in intraalveolar fibrinous deposits in inflammatory and other conditions of the lung associated with significant transudation of plasma into the air spaces. Our results further indicate that thromboplastic activity of lung ext’ract’s as judged by substituting lyophilized or nonlyophilized lung extracts for brain thromboplastin in the one stage prothrombin time technique, increases in parallel with the surface activity of these extracts, being maximal, 16 (’ 1) seconds, in the strongly surface active sediment B. The above modified one stage prothrombin time technique, therefore, can be used as an additional parameter to be correlated with other criteria relating to surfactant properties. Since lung surfactant is a thromboplastic substance which may conceivably enter the circulation following lung trauma or disease, an invest,igation was recently undertaken to evaluate possible syst’emic effects of intravascular surfactant leakage. Preliminary results indicated that intravenous or left intracardial injection of surfactant lipoprotein results in pulmonary microthrombosis in association with endothelial damage, especially in animals pretreated with steroids (Rappaport et al., 1970). In view of the above findings it is worth further exploring the possibility that lung damage in various disease processes may predispose to the development of local and systemic thromboembolic phenomena. C. Reversible versus irreversible foms of swfactant inactivation It has been suggested that following interaction of fibrinogen with surfactant lipoprotein the latter is inactivated due to formation of a new substance (Taylor and Abrams, 1966). Our findings, however, indicate t’hat, the interaction of serum with surfactant results in a similar type of surfactant inactivation which should be considered as a reversible reaction since a surface active sediment B, rich in phospholipids, can be recovered from the above seemingly inactive mixture. By contrast, interact’ion of surfactant with plasma results in an irreversible form of surfactant inactivation due to clot formation. Our results further indicated t’hat the clot incorporates both surfactant phospholipids and cells, thus depleting the washing-plasma mixt’ure of its surfactant content and cellular elements. The above coagulative form of surfactant depletion may represent a mechanism of surfactant deficiency in lung diseases characterized by fibrinogen-rich alveolar exudate and formation of hyaline membranes (i.e., hyaline membrane disease of the newborn, primary atypical pneumonia, lung damage following oxygen toxicity, etc.). On the other hand, the seemingly reversible form of surfactant damage in the lung washing-serum mixture may occur in edematous conditions of the lung. This interpretation explains our previous observat’ion that inactive lung extracts from edematous dog lung following unilateral pulmonary artery ligation yield a strongly surface active sediment B (Balis et al., 1969). It is known that in lungs of infants with hyaline membrane disease (HMD),
NORMAL
AND INACTIVATED
LUNG
SURFACTANT
261
alveolar macrophages are inconspicuous during the first 24 hours after delivery. There is also evidence that in addition to cell debris and plasma proteins (Balk et al., 1966) human hyaline membranes contain surfactant (Craig, 1964). On the basis of the present in vitro studies we propose that in HMD both surfactant and macrophages are incorporated in the intraalveolar fibrinous deposits which are eventually transformed into hyaline membranes following disintegration of the cellular elements. Further histochemical, ultrast’ructural, and biochemical studies are in progress to evaluate and compare the mechanism of membrane formation in in vitro models and in various human and experimental lung diseases. ACKNOWLEDGMENT The technical assistance of Elaine Smith, Maria Mezari, Binnie Lambert, is gratefully acknowledged. REFERENCES Surface phenomena
and Donna Janota
AVERY, M. E.. AND SAID, S. (1965). in lungs in health and disease. &Zeditine 44,503-526. BALIS, J. U., AND CONEN, P. E. (1964). The role of alveolar inclusion bodies in the developing lung. Lab. Invest. 13,1215-1229. BALIS, J. U., DELIVORIA, M., ANDCONEN, P. E. (1966). Maturation of postnatal human lung and the idiopathic respiratory distress syndrome. Lab. Invest. 15, 530-546. BALIS, J. U., AND &PPAPORT, E. S. (1969). In vitro inactivation of pulmonary surfactant and ‘(hyaline membrane” formation. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 26,745. BALIS, J. U., RAPPAPORT,E. S., PIFARRE, R., AND NEVILLE, W. E. (1969). Ultrastructural and surf&ant changes of the dog lung following pulmonary artery ligation. Lab. Invest. 20,574. BALIS, J. U., SHELLEY, S. A., MC&E, M. J., AND RAPPAPORT,E. S. (1970). Ultrastructure and quantitation of normal and in vitro inactivated lung surfactant. Amer. J. Pathol. 59, 95a. BENSCH. K., SCHAEFER, K., AND AVERY, M. E. (1964). Granular pneumonocytes: Electron microscopic evidence of their exocrinic function. Science 145,1318-1319. in the BERNSTEIN, J., YANG, S. S., HAHN, H. S., AND KIHECAWA, Y. (1969). Mucopolysaccharide pulmonary alveolus. I. Histochemical observations on the development of the alveolar lining layer. Lab. Invest. 21,420-425. BONDURANT,S., AND MILLER, D. A. (1962). A method for producing surface active extracts of mammalian lungs. J. Appl. Physiol. 17, 167-168. BUCKIXGHAM, S., HEINEMANN, H. O., SOMMERS,S. C., AND MCNARY, W. F. (1966). Phospholipid synthesis in the large pulmonary alveolar cell. Amer. J. Pathol. 48,1027-1041. CAVAGNA,G. A., VELASQUEZ, B. J., WETTON, R., AND DUBOIS, A. B. (1967). Cellular absorption of pulmonary surface-active material. J. Appl. Physiol. 22, 98%989. CLEMENTS, J. A., AND TIERNEY, D. F. (1965). Alveolar instability associated with altered surface tension. In “Handbook of Physiol.” (W. 0. Fenn and H. Rahn, eds.), Vol. 2, sec. 3. Amer. Physiol. Sot., Washington, D.C. CRAIG, J. (1964). The distribution of surface active material in the lungs of infants with and without respiratory distress. Biol. Neonatorum 7, 185-202. FINLEY, T. M., PRATT, S. A., LADMAN, A. J., BREWER, L., AND MCKAY, M. B. (1968). Morphological and lipid analysis of the alveolar lining material in dog lung. J. Lipid Res. 9, 357-365. FOLCH, J., LEES, M., AND SLOANE STANLEY, G. H. (1957). A sample method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-509. GLADSTON, M., SHAH, D. O., AND SHINOWARA. Y. (1969). Isolation and characterization of a lung lipoprotein surfactant. J. Colloid Interface Sci. 29,319-334. GIAMMONA, S. T., KERNER, D.. AND BONDURANT, S. (1965). Effect of oxygen breathing at atmospheric pressure on pulmonary surfactant. J. AppZ. Physiol. 20, 855-858. GREENFIELD, L. J., AND DUNCAN, R. C. (1968). Quantitative dynamic pulmonary surfactant. determinations with the pneumatic surfactometer. Surgery 64,351-358.
BALIS GRONIOWSKI,
lungs.
Nature
J.,
AND
204,
BICZYSKOWA,
W.
(1964).
ET
AL. Structure
of
the
alveolar
lining
film
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