BRONCHO-PULMONARY PROTECTIVE COMPLEX WITH COMPLETE SET OF SURFACTANT-ASSOCIATED PROTEINS

BRONCHO-PULMONARY PROTECTIVE COMPLEX WITH COMPLETE SET OF SURFACTANT-ASSOCIATED PROTEINS

Martin van Eijk, Sigurd Krieger, Alersey N. Savchuk

Affiliation of the authors:

¹ Crimea State Medical University, Deptartment of Pathology, Bul.Lenin 5/7, Simferopol, 95006, Ukraine
² Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine Utrecht University, NL-3508 TD Utrecht, The Netherlands
³ Medical University Vienna, Department of Clinical Pathology, Waehringer Guertel 18-20, 1090 Vienna, Austria
⁴ Department of Structure and Functions of Proteins,
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine 01601, Kiev, Leontovichstr. 9, Ukraine

Abstract word count: 190

Conventional surfactant agents used for surfactant-replacement therapies provoke unwanted side effects by contaminants introduced during their preparation process. We established a new technology for the preparation of broncho-alveolar lavage of pig lungs without lesions of pulmonary structures.

We used a modified "Donders" device for the lavage preparation that gave us more control over the pressure progression by passive aspiration of the lavage fluid. The remaining tissue was controlled for damages by light and electron microscopy. Biochemical techniques were used for the analysis of the resulting lavage product.

The obtained substance was called broncho-alveolar protective complex (BAPC) and contained the major components of local protection system of the lung like IgG, a 1-antitrypsin, important phospholipids of endogenous surfactant and surfactant-associated proteins SP-A, SP-B, SP-C, SP-D. As confirmed by light and electron microscopy no damage or ruptures of interalveolar septa or signs of alveoli over distension were observed.

Since BAPC resembles a more perfect composition of the natural compounds of the lung protection system, further studies about its biological activity in-vivo and in-vitro should form the basis for the creation of new surfactant-containing drugs for treatment of broncho-pulmonary diseases.

For nearly two decades the surfactant-replacement therapy of broncho-pulmonary diseases is based on the application of preparations of exogenous surfactants of various classes and types. The most frequently used surfactants in clinical practice are natural and natural modified surfactants. Unfortunately, none of these surfactant preparations produced today contains a complete set of surfactant-associated proteins. In such preparations SP-B and SP-C are present in various quantities [4,8], but a considerable negative effect on the efficiency of such preparations is produced by absence of SP-A and SP-D [10].

The main reason for the inefficiency of such preparations relies on technological problems by using homogenate of animal lungs as initial raw material. Apart from the components of surfactant, a great number of plasma and tissue proteins are inevitably brought into the extract. This also occurs at pumping lavage fluid into the airways under practically uncontrollable pressure and results in disruption of interalveolar septa and thus leads to contamination with proteins of blood plasma. At presently there are no medical agents, including exogenous surfactants, which produce an effect similar to that of endogenous surfactant and moreover would perform the function of the local protection system of the lungs.

Our main task was to create conditions that allow receiving broncho-alveolar lavage without any damage of epithelial and other structures of bronchi, bronchioles and alveoli. Besides, lavage should be carried out by the application of a suitable fluid which would dissolve all the most important substances mentioned above. With the purpose to prevent these undesirable admixtures to the extraction phase, a technique was developed to ensure the presence of only a small amount of

SP-B and SP-C in the final product associated with phospholipids of the surfactant and in presence of SP-A and SP-D.

The lungs of healthy pigs were used. The animals were sacrificed and the heart-trachea-lung complex was prepared. The heart was separated, trachea and lungs were enclosed into a special container with constant temperature of 37°C and taken to the laboratory. The investigation started in 2-3 hours after isolation of the organs. Only lungs without signs of foam presence in trachea, main bronchi, features of visible ruptures of visceral pleura and subpleural hemorrhages were used for lavage preparation.

Based on a well known physiology model of the thoracic cavity we used the device for the study of thorax pressure according to Donders, extended by a number of important features. The intubation-tube was inserted into the trachea and the outlet-end was plunged into a reservoir filled with chloroform (250-300 ml). Then the intubated lung was placed in a hermetic chamber, in which by means of a pump the positive or negative pressure corresponding to physiological parameters was consistently maintained.

The process for receiving broncho-alveolar lavage consisted of three phases.

During the first phase an overpressure of about 765-770 mmHg was created in the chamber, which resulted in compression of the lung and extrusion of predominantly all of the residual air from the lung airways and alveoli. This was confirmed by appearance of air bubbles from the intubation tube plunged into chloroform. As soon as excretion of bubbles stopped, pressurization of the chamber was ceased in order to prevent mechanical damage of interalveolar-septa and bronchiolo-bronchial walls. The absence of such damages is testified by the results of morphological analysis (see below).

The pressure in the chamber was gradually lowered, first up to atmospheric level and then up to nearly 750 mmHg," this corresponds to physiological parameters of intrapleural pressure on the peak of inspiration. During this phase chloroform through intubation tube without any additional efforts filled in airways and free alveolar spaces.

During the third phase the chamber was set again under pressure corresponding to parameters of the first phase that was accompanied by removal of chloroform from alveoli and airways through intubation tube. This technology allowed recovering up to 50 % of the lavage fluid introduced into the lung. Lavage by the described method was carried out only once per lung.

By morphological studies (see bellow) we established that a short-term contact (0.5-1 min) with chloroform was not accompanied by any damages of cellular membranes and cells of bronchial, bronchiolar and alveolar epithelium and prevented the entry of serum and tissue proteins into lavage fluid.

Preparation of the broncho-alveolar protective complex (BAPC)

The received chloroform solution was filtered through paper filters for removing admixtures of mucus and cellular elements and dried up under vacuum in presence of liquid nitrogen. Then by ultrasound cavitation an emulsion was prepared in physiological saline (0.9 % NaCl) to maintain 75 mg of phospholipids of lung surfactant per 1 ml of saline. We named the substrate obtained by this procedure the broncho-alveolar protective complex (BAPC).

With the purpose to show the diffusion of lavage-fluid within the lung we used physiologic saline stained with ink to lavage the lungs in accordance to the specified conditions. After each of the described phases slices were cut out from the lungs for further histological analysis. Thus each time new samples of the lungs were used. The same procedure was carried out by lung lavage with chloroform solution. Further, the samples were taken from lungs lavaged with chloroform under pressure until chloroform began to penetrate outward the lungs and flow down the surface of visceral pleura - thus the common standard procedure of lung lavage was copied. Light and transmission electron microscopy with preparation of material according to standard procedures were used.

Biochemical analysis of BAPC

Finally, the received BAPC was investigated by a series of biochemical methods. With the purpose to identify the protein composition of BAPC emulsion, the material was investigated by disc-electrophoresis with the use of gels from 8 up to 15% (T) [14,15]. The analysis of electropherogramswas carried out with the use of the software kit Image Master Total Lab v.2.01. Besides, the samples were analyzed by sodium-dodecyl-sulfate polyacrylamide electrophoresis in Laemmli system, different concentrations of bovine serum albumin in amounts of 10, 50, 100 and 1000 ng as reference

were loaded with further staining of the gels by silver stain according to Blum et al. Western blotting was used to determine the presence of surfactant-associated proteins.

To determine the lipid-phospholipid structure of BAPC the method of thin-layer chromatography was applied with the use of standards of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylglycerin. Percentage of phospholipids was calculated with the half-quatitative method of spots planimetry.

The morphological analysis by light microscopy testified the fact that on reception of chloroform lavage according to the described technology was not accompanied by any damages of pulmonary structure in contrast to the usual technique of pumping the liquid into the lung airways. No wall ruptures of interalveolar septa or signs of alveoli overexpansion, was observed, nor interstitial or intraalveolar edema were observed. Futher, no increased permeability of capillary walls of the interalveolar septa or damage of the vascular walls occurred. This was indicated by the absence of erythrocytes and other corpuscular elements of blood in the lumen of alveoli (Fig.l a-c). By electron microscopy it was demonstrated that epithelial cells of the respiratory part of the lungs as well as bronchioles and bronchi appeared inconspicuous, the continuity of the epithelial lining was kept uninterrupted and no lesions from the basal membrane and capillary walls-were -found (Fig.2). These findings proove that the developed technology allowed getting lavage from the internal broncho-bronchiolo-alveolar surface without any damage of the pulmonary structures. These facts led us to presume that BAPC includes only components that are located at the internal surface of the lung.

Thin-layer-chromatography (Fig.3) of the investigated samples revealed spots corresponding to neutral lipids, cholesterol and its ethers, complex of fatty acids, as well as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin and traces of lysophosphatidylcholine (Table 1). This implies that BAPC contains all basic lipid-phospholipid components, which are well known components of endogenous surfactant. Many of these components are included in the composition of preparations of natural and natural modified exogenous surfactants. An additional spot was found by thin-layer-chromatography suggesting the presence of small amounts of mono- and diglycerides. However, this assumption requires further studies. Protein molecular weights revealed by staining-techniques and western-blotting corresponded to components that are known to be included in the structure of the local protection system of the lung (Table 2). In particular, by western-blotting we identified the presence of aggregated forms of SP-A (Fig.4). Unfortunately SP-D detection was not successful because its concentration was below the detection limit (5 ug/ml). However, given the fact that SP-A is a lipid

- associated protein and SP-D is a soluble protein and the fact that amounts of SP-D in the lung are about 10-fold lower than that of SP-A it is reasonable to assume that SP-D levels are very low.

Analysis of BAPC samples by disc-electrophoresis in Laemmli system showed that among the spots they contain there are spots with molecular weight from 4-5, 8, 35, 65 to 103, 111, 119 kDa (Fig.5) and even 127, 139 and 158 kDa. The investigation of other samples using a silver stain system according to Blum (Fig.6) once again demonstrated the presence among others the bands with molecular weights about 62 kDa (band 3), 32 kDa (band 4) and 5,5 kDa (band 5).

Besides electrophoretic analysis in presence of appropriate reference to secretory IgA revealed the absence of this protein in BAPC structure. Finally, our data allowed us to assume the presence of small concentrations of al-antitrypsin in the composition of the investigated samples.

It is well known that no commonly used surfactant preparation has all characteristics of human endogenous surfactant. So their effectiveness leaves much to be desired. We consider that this is connected with two circumstances.

First, extraction of surfactant from the local protection complex inevitably changes its functional properties that undoubtedly has a negative effect on its clinical using.

Secondly, moden techniques of preparation of exogenous surfactants allow in a vast majority of cases, of containing only two from the four surfactant-associated proteins, namely SP-B and SP-C, that do not exert immune modulatory properties.

Our newly developed technology allows of receiving a complex mixture that exhibits the characteristics encompassing the composition of the local protection system of the lung. Presented in a form of emulsion, this substance, in particular, contains a protein-lipide complex with all known proteins of endogenous surfactant (SP-A, SP-B, SP-C and, probably, SP-D), as well as such important components of local protection system of the lungs as IgG and, probably, al-antitrypsin. Non-identified protein components of BAPC in the molecular weight range of 100 - 120 kDa are apparently protein-lipid-carbohydrate and have to be identified in ongoing work. Finally, our data enable us to carry out further studies to proove the biological activity of BAPC in vitro and in vivo.

1. Turner-Warwck M. Immunology of the iung. Edward Arnold; 1978.

2. Clare J, Reid KBM, Sim RB. Collectins and innate smmunity in the lung. Microbes Infect 2000;2(3):273-278.

3. Hawgood S, Poulin FR. The pulmonary collectins and surfactant metabolism. Annu Rev Physiol 2001;63:495-519.

4. Possmayer F, Rodrigues-Capote K, Ney K, Manzanarez D. Role of Surfactant Apoproteins in Surfactant Function. Appl Cardiopulm Pathophysiol 2004;13(l):75-77.

5. Battenburg JJ. Surfactant proteins SP-A and SP-D: Interactions with patogenesis. Appl Cardiopulm Pathophysiol 2004; 13(1): 14-16.

6. Crough E, Wright JR. Surfactant proteins A and D and pulmonary host defense. Annu Rev Physiol 2000;63:521-554.

7. Haagsman HP, van Eijk M, Yerias MV. Porcine collections SP-A and SP-D: Expression sites, Structure and Function. Appl Cardiopulm Pathophysiol 2004;13(l):38-39.

8. McCormack FX, Whitsett JA. The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J clin Invest 2002; 109:707-712.

9. Wchite CW, Dreene KE, Alles CB, Shennon JM. Elevated expression of surfactant proteins in newborn rets during adaptation to hyperoxia. Am J Respir Cell Mol Biol 2001;25(1):51-59.

10. Wright JR. Regulation of immune cell function by surfactant prjteins. Appl Cardiopulm Pathophysiol 2000;9(3):314-315.

11. Cochraine PG. Critical Examination of the Role of SP-B in Alveolar Expansion. Appl Cardiopulm Pathophysiol 2004;13(l):27-29.

12. Perez-Gil J. Lipid-protein interactions of hydrofobic proteins SP-B and SP-C in lung surfactant assembly and dynamics. Pediatr Pathol Mol Med 2001;20(6):445-469.

13. Robertson B, Taeusch HW. Surfactant Therapy for Lung Disease. Nev-York: Marsel Dekker; 1995.

14. Affinity Chromatography. Principles and Methods. Handsbooks from Amershem Pharma Biotech; 2001.

15. Gel Filtration. Principles and Methods. Handsbooks from Amershem Pharma Biotech; 2001.

 

Figure legends

Figure.1.

Figure.1.a.

Figure.1.b.

Figure.1.c.

Figure.2.

Figure.3.

Figure.4.

Figure.5.

Figure.6.

N	Lipid/phospholipid of BAPS	Percentage
1	Mono- and diglycerides	1,525±1,076*	M-DG
2	Neutral lipids	6,775±0,580	NL
3	Cholesterol	10,50±5,416	Ch
4	Eters of cholesterol	9,475±1,639	Ech
5	Phoshatidylethanolamine	10,20±2,643	PhE
6	Fatty acids	34,65±5,479	FA
7	Phosphtidylglicerol	4,675±1,495	PhG
8	Phosphtidylgcholine	13,60±1,629	PhC
9	Phosphtidylinositol	5,025±0,296	PhI
10	Sphingomyeline	2,55±0,292	Sph
11	Lysophosphatidylcholine	0,775±0,521*	Lys

* - the fractions were developed not in all samples of BAPC

Components of system of local protection of lungs	Molecular mass (kDа) according to literature data	Molecular mass in BAPC (kDа)	Identification in BAPC and quantity in microgram/ml
Secretory Ig A	45	not present	not present
Ig G	~150	150	Ig G 0.030
α1-antitrypsin	55-58	57	α1-antitrypsin 0.560
SP-A	30-35	31-35	SP-A* 0.032
SP-B	~8	8	SP-B* 0.180
SP-C	~4	4	SP-C* 0.005
SP-D	55-65	57-65	SP-D 0.005
		100-110	non-identified components 1.110

* - availability is confirmed by Western-blot method. Total: 1.922

A, B –Pig lungs after pumping of the lavaged fluid in the airways: there are small intaalveolar hemorrhages (dark arrows) and ruptures of interalveolar walls (light arrows). H&E. A – mag. 100; B – mag.400.

C – Pig lungs after described technique of soft chloroform lavage. There are no wall ruptures of interalveolar septa, signs of alveoli over distension and intraalveolar hemorrhages. H&E. Mag. 100.

Respiratory unit of the pig lungs after soft chloroform lavage according to the described technique.

There are no lesions of alveolocites of the 1-st type (A1), of epithelial lining (dark arrows) and capillaries’ walls (Cap). Only alveolar macrophages (AM) and surfactant debris and particles (Light arrows) are seen into the alveolar lumen. Electron microphoto. Mag.3000.

The scan of thin layer chromatogram with developing of pilid/phospholipid components of BAPS

Mono- and diglycerides – M-DG; Neutral lipids – NL; Cholesterol – Ch; Eters of cholesterol – Ech; Phoshatidylethanolamine – PhE; Fatty acids – FA; Phosphtidylglicerol – PhG; Phosphtidylgcholine – PhC; Phosphtidylinositol – PhI; Sphingomyeline – Sph; Lysophosphatidylcholine – Lys.

The left blot shows dimeric SP-A in BAPL samples ( in the 3 left lines; the first lane shows a faint band, but SP-A is definitely present in this sample). To the rught are samples of puure SP-A (mostly monomeric) and molecular markers.

The right blot shows that SP-D is not present (left three lanes); to the right samples of pure SP-D and molecular markers.

Scan of disc-electrophoresis of BAPC samples (4 and 4.1) and albumine (67kDa), streptokinase (47kDa), insuline (57kDa), -lactalbumin (14,2 kDa), -lactalbumin (18 kDa).

Scan of electrophoresis of BAPC samples with constant identified bands with molecular weights 62 kDa (band 3), 32 kDa (band 4) and 5.5 kDa (band 5).

 

25.02.2010