Effect of bovine seminal ribonuclease (BS-RNase) on pigs bone marrow cells

The effect of bovine seminal ribonuclease (BS RNase) on bone marrow cells in miniature pigs was studied. BS RNase at the concentration 20 and lOOug/ml preincubated 1 h with bone marrow cells from normal miniature pigs did not influence the formation of CFU-GM and BFU-E colonies cultivated in methylcellulose medium. The bone marrow cells kept in 4° C survived better in the presence of BS RNase than control cells cocultivated with bovine serum albumin (BSA). There was proved that pig bone marrow cells are able to bind the BS RNase on their surface without any morphological damage. For the experimental bone marrow transplantation in miniature pigs the whole body irradiation of 8G with a cobalt source was used. Two recipients from seven accepted semi-incompatible bone marrow cells preincubated 2 h with BS RNase (10mg/5xl0 cells). No recipient from eight transplanted control animals accepted semi-incompatible bone marrow cells preincubated with BSA.


Introduction
Spermatozoa are foreign cells for reproduetive organs of females.They must be protected for a certain time against female immune cells and production of antibodies.This protection probably occures in both sexes: male and female.Numerous studies showed that human and animal seminal plasma and spermatozoa includes many immunosuppressive substances (SHIVAJI et al., 1990).The bovine seminal ribonuclease (BS-RNase) is an antitumor and immunosuppressive substance which was determined in the bull seminal plasma (MATOUSEK, 1985;D'ALESSIO et al., 1991) and using an ELISA method in a very large amount (in average 1132 (ig/ml -MATOUSEK and KLAUDY, 1998).The biological properties of BS-RNase were accounted to its dimeric form, since this structure is unique among the vertebrate RNase superfamily (D'ALESSIO et al., 1991).The DNA sequence of the gene encoding BS-RNase was determined and recombinant preparation of BS-RNase was synthetized (KIM and RAINES, 1993;DE NIGRIS et al., 1993).We have found that the biological activities, including the immunosuppressive effect, are identical with these of the native BS-RNase (KIM et al., 1995).The potent immunosuppressive activity of this enzyme and its favourable effect on hemopoietic cells in animal and human bone marrow cultures (STANEK et al., 1978(STANEK et al., , 1982;;SOUCEK et al., 1981SOUCEK et al., , 1986SOUCEK et al., , 1996;;TAMBURINI et al., 1990) urged us to examine BS-RNase effect on experimental bone marrow transplantation in semi-incompatible miniature pigs.

Animals
Miniature pigs (Minnesota type), 6-15 kg weight and 3-5 months old, were selected for the lymphocytes, bone marrow cells and transplantation experiments.All animals were bred at our Animal Centre.Pigs from single letters were typed for the blood groups and histocompatible antigens 14-20 days before transplantation.The selected pigs were penned at least 4 days prior to bone marrow transplantation and during posttransplantation time individually.The experimental house was sterilized by germicide lamps before the pig's housing.The animals were systematically deparasitized.

Ribonucleases and other substances
Bovine seminal ribonuclease (BS-RNase) was isolated from bull seminal plasma by precipitation with acetic acid and amonium sulphate and the supematant subjected to CM Sephadex C-50 and Sephadex G-100 column chromatography (DOSTÄL and MATOUSEK, 1973).Bovine pancreatic ribonuclease (RNase A) was obtained from ICN Biomedicals Inc., USA.Phytohemagglutinin (PHA) was obtained from Wellcome Diagnostics, England.Swine anti-rabbit IgG/FITC was produced by USOL, Prague, Czech Republic.Anti-BS-RNase antibodies were produced by reimmunization of two rabbits and IgG isolated.Verograftn (produced by Leciva, Prague) was used for isolation of lymphopcytes.The bovine serum albumin (BSA) was obtained from the Institute of Sera and Vaccines, Prague.RPMI 1640 and MEM medium were obtained from the Institute of Haematology and Blood Transfusion, Prague.

Estimation of Colony-Forming Activity
Bone marrow cells obtained by a puncture of vertebral bodies and ribs of fresh killed pigs were suspended in Iscove medium mixed 1:1 with ACD and supplemented with 10% of FCS, antibiotics and heparin (20 IU/ml).Colony-forming activity of hematopoietic pig cells was carried out aecording to the routine assay for human hematopoietic progenitor cells (SUTHERLAND et al., 1991) on methylcellulose medium MethoCult GF H4434.The effect of BS RNase on the growth of erythrocyte colonies (CFU-E and BFU-E) and of granulocytes (CFU-GM) was estimated as follows: 0.1 ml of bone marrow cells Suspension (4 x 10 6 /ml) was preincubated for 1 h with two concentrations of BS RNase (100 and 20 ug/ml).The control samples were preincubated with 0.1 ml of PBS.The pretreated cells were diluted in cultivation medium, then mixed with the methylcellulose medium to the final concentration 10 x 10 cells/ml and cultivated in Petri dishes (Falcon cp 35 mm) for 14 days at 37°C under a humidified atmosphere containing 5% C0 2 .The development of erythrocyte colonies (BFU-E) and granulocyte colonies (CFU-GM) were counted over the whole dish at 60x magnification in a binocular microscope.

Indirect immunofluorescence
Rabbit IgG was isolated from the serum of two rabbits immunized by three series of injections (one set 4 injections of 10 mg of BS-RNase during 4 weeks) and used as an antibody to BS-RNase.Pig bone marrow cells were suspended in PBS, pH 7.2 at the concentration 10 x 10 6 /ml.The cell Suspension was ineubated with 100 ug/ml BS-RNase Solution 1:1 at 37° C for 60 min.After ineubation and three washing with PBS, the cell Suspension was spread on the slides, fixed with methanol, washed three times with PBS and rabbit IgG carrying anti-BS-RNase antibodies was added.Rabbit IgG bound to cell surface-associated BS-RNase was then detected by fluoresceinconjugated swine anti-rabbit globulin (Institute of Sera and Vaccines, Prague).The control cells were ineubated with normal serum IgG instead of BS RNase one.

Survival of pig bone marrow cells in BS-RNase Suspension
1 x IO 8 pig bone marrow cells were suspended in 10 ml MEM medium supplemented with 10% fetal calf serum and 5 mg of BS-RNase.This Suspension was kept in a refrigerator (4°C) for 5 days and the viability of cells was estimated by trypan blue exclusion test (Sigma, St. Louis, USA) after 1, 24, 48, 72, 96 and 120 h.The control samples contained 5 mg of BSA instead of BS-RNase.Survival of bone marrow cells .was estimated in 200 cells counted.

Electron microscopy
Bone marrow cells studied for survival experiments ineubated 48 h with BS-RNase were centrifuged, fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 60 min., washed with 0.1 M phosphate buffer and refixed in 1 % osmium tetroxide in 0.1 M phosphate buffer for an additional 60 min.After washing with the buffer and dehydration in graded alcohol and acetone, the specimen was embedded in Durcupan ACM Fluka.Thin sections were cut on an LKB III ultramicrotome, stained with uranyl acetate and lead citrate, using a modification of the method (VENABLE and COGGESHALL, 1965) and examined in a JEM7A electron microscope at 80 kV.

Antigenic characterization of donors and reeipients
The blood group and histocompatibility phenogroups were tested in all miniature pigs used as donors and reeipients of bone marrow cells.Blood group antigens were serologically tested by 74 monospeeifie reagents by means of the direct agglutination test, antiglobullin (Coombs) test, dextran and haemolytic tests (JUNEJA and VÖGELI, 1997;HOJNY and HRADECKY, 1971).The corresponding 73 phenogroups have been controlled from 16 locusses (JUNEJA and VÖGELI, 1997).Histokompatibility antigens (in pigs SLA, SLB and SLC Systems) belong to class I antigens (LUNNEY and BUTTER, 1997) and the corresponding SLA-A typing alloantisera have been developed mostly by repeated isoimmunization procedures in miniature pigs; the last international comparative test was reported by RENARD et al. (1988).The experimental pigs were tested by 6 Standard antisera designated by W and 10 antisera specifically prepared by miniature pig immunization and designated the letter L (RENARD et al., 1988).SLB and SLC antigens are independent on SLA genes and there are genetically controlled in locusses designated B and C (HRUBAN et al., 1983(HRUBAN et al., , 1988)).These antigens are on pig leukocytes but not on erythrocytes.Seven SLB antigens are genetically controlled by 13 alleles (HRUBAN et al., 1988) two SLC antigens are genetically determined by 2 alleles and this system is closed (HRUBAN et al., 1983).SLA, SLB and SLC antigens were determined at least by two different reagents.

Irradiation procedure
Nonanesthetized, fastened miniature pigs were irradiated using Symmetrie bilateral cobalt -60 sources at the Medical Faculty, Hradec Krälove.A single dose of 8 Grays total-body irradiation was given at 20 rads/min (SAKAMOTO et al., 1988).The bone marrow cells from a selected donor were infused into reeipients 12-16 h after irradiation.

Bone marrow transplantation
The allogenic donor was anesthetized, exsanguinated and bone marrow harvested surgically from the humeruses, tibias and femures.Vertebral bodies and ribs were melted by a mincing mashine under aseptic conditions.Bone marrow cells were suspended in RPMI 1640 and ineubated at room temperature.Suspensions were filtered through 100 mesh nylon gauze and centrifuged at 1200 rpm for 15 min.The cell pellet was 3 times washed and resuspended in RPMI 1640 medium supplemented with 5 % normal pig serum, 100 U/ml penicilin, and 100 ug/ml of streptomyein.The bone marrow cell Suspension was infused in a volume of 450 to 600 ml into a reeipient (5 x IO 8 cells/kg) about 3 h after sampling.Seven irradiated reeipients were infused with the Suspension preincubated 2 h with BS-RNase (10 mg/5 x IO 8 cells) while 8 control reeipients received bone marrow preincubated with BSA (bovine serum albumin).
Posttransplantation care Penicilin 400.000I.U.and Streptomyein 400 mg i.m. was injected daily starting on day 1.Biseptol 12 mg/day into fodder, Supristol 120 mg/day and 10% glukose were added into the drink from the first day after the transplantation and continued through day 16.Fresh whole blood from the donor (200-300 ml) was given at day 7 as a source of platelets.Complete blood counts of reeipients were examined daily and animals surviving more than 16 days were typed for blood group and histocompatible antigens.

Statistical analysis
Results are presented as mean ± Standard error of mean (SEM).The data were analysed statistically using Fisher's t-test.

Effect of BS-RNase on colony-forming activity
There were relatively large differences in the colony-forming activity of pig bone marrow cells between the single pigs.These individual differences were not influenced by the presence of BS-RNase in the cultivation medium.Both tested quantity of BS-RNase in the medium (20 and 100 ug/ml) did not change substantially the growth of granulocyte and erythrocyte colonies (Table 1).

Binding of BS-RNase by pig bone marrow cells
The binding of BS-RNase on the surface of bone marrow cells from 6 miniature pigs are shown in Figure 1 and Table 2.These cells were without any harmful effect (Fig. 2).Neither erythrocytes or unstimulated lymphocytes absorbed BS-RNase (data not shown).Influence of BS-RNase on the survival of bone marrow cells Bone marrow cells (1 x 10 8 ) from 5 donors suspended in 10 ml of MEM medium with 5 mg of BS-RNase and kept at 4°C survived better in comparison to cells treated with 5 mg of BSA, (Table 2).After 96 h storing 31 per cent of dead cells in the MEM medium with BS-RNase and 91 per cent in the medium with BSA were determined and after 120 h the values of dead cells were 42 and 94 per cent.

Antigenic differences between reeipients and donors
Fifteen reeipients were transplanted by the bone marrow cells ofthe 5 donors selected mostly from the same litter and the same sexes (Table 3).Only three reeipients were transplanted by the bone marrow cells identical in SLA, SLB and SLC haplotypes with a donor, the other reeipients obtained bone marrow cells that differed in histocompatibility haplotypes (Table 3).

Haematological changes
The irradiation exerted a profound leucopoenia in all animals.After a rapid fall after the irradiation the decrease in leueocyte counts continued up to 10-14 days.Number of leueocytes 12 x 10 3 /mm 3 before irradiation decreased to 7 x IO 2 /mm 3 in two weeks after the transplantation.The number of erythrocytes did not change so much as that of leueocytes (6 x IO 6 to 3-5 x 10 6 ) after the transplantation.In the pig no 4 that survived more than 2 years the number of lymphocytes normalized after 50-60 and erythrocytes after 30 to 40 days.

Disease and mortality
Reeipients after irradiation and transplantation suffered from haemorrhage, crusting pneumonia and severe infections.Some animals were killed in a State of sever cachexia.Occasional hyperthermic periods, diarhea and bleeding were also observed in spite of antibiotic therapy.

Anti-SLA, SLB and SLC antibody production in transplanted pigs
The appearance of leueocytotoxie antibodies was looked for almost in all of the serum samples drawn during the experimental period.Specificity studies on a panel of cells have shown that cytotoxic activity is directed against leueocyte antigens.The titres of antibodies declined from 0 to 1 : 4 both in reeipients transplanted with cells ineubated with BS-RNase or without this enzyme.

Transplantation results
Three reeipients (no 1, 3, 39) that received compatible bone marrow cells in SLA, SLB and SLC antigens from their sister and brother (Table 3) died 12, 15 and 17 days after transplantation.The donor's cells for the reeipient 39 were preincubated with BS-RNase, and the reeipients no 1 and 3 by BSA.The crusting in skin, lung, intestines and liver was observed after the death during autopsy as nose bleeding in last three days of life in reeipients 1 and 3.The animal 39 died of systemic infection without any crusting in the skin and body organs, but with the decreased number of leueocytes 8 5 0/mm 3 and erythrocytes 3 x 10 6 /mm 3 .The next reeipients 4, 38, 44, 57, 81 and 48 received bone marrow cells from SLA semi-incompatible genotyped donors (Table 3) treated for 2 h with BS-RNase.The pigs 4 and 57 that differed from the donors ofthe same litter in L2.8 and L2.7,3.4,2antigens accepted the donor bone marrow cells.These animals remained alive up to 26 months when the reeipient 4 was killed and up to 33 days when the reeipient 57 died.
The number of leueocytes in the reeipient 4 decreased from 4.400 before transplantation up to 600 on the 7 day after the transplantation.The erythrocytes decreased from 7,2 x IO 6 up to 3,2 x 10 .The number ofthe blood cells decreased in the reeipient 57 from 5.100 up to 920 of leueocytes and from 6,6 x IO 6 up to 5,9 x 10 of erythrocytes per 1 mm 3 of blood.During the next 15 days (22 days after transplantation) the number of blood cells increased potently in the pig 4 to 6.800 of (leueocytes) and 3,930.000 of (erythrocytes) and in the pig no 57 to 4.010 leueocytes.The erythrocytes of this pig decreased into 5,3 x 10 6 /mm 3 .In this time all supported medicaments were removed, and these two reeipients transported into the common stable with other pigs.By means of analysis of histocompatibility and blood group antigens of these two reeipients was found that they obtained 50-90 per cent of erythrocytes and 100 and 80 per cent of leueocytes from their donors.The following 7 and 2 (in the pig 57) Screenings of antigens in the reeipients confirmed again the presence of donor antigens in their blood.
The next reeipients no 38, 44, 81 and 48 that were transplanted by donor's semi-in compattble bone marrow cells ineubated with BS-RNase died between 9-16 days after the transplantation.Haemorrhage in skin, intestines, lung, stomach and haemoglobin in the urine were diagnosed in them.
The reeipients transplanted by donor's semi-incompatible bone marrow cells ineubated instead of BS-RNase by BSA (no 58,59,92,42,48 and 49) died between 8 and 14 days.The number of leueocytes decreased 7 days after the transplantation in the reeipient no 42 up to 400 and number of erythrocytes up to 1,010.000 in 1 mm 3 of blood.In the other reeipients transplanted by donor's cells ineubated with BSA the number of blood cells moved similarly as in pigs transplanted by bone marrow cells ineubated with BS-RNase.All reeipients of this group suffered by haemorrhage on skin, intestines, some of them on liver and stomach, haemoglobin in urine and bleeding from noses.
Discussion Experiments with miniature pigs were performed to establish the effect ofthe bovine seminal ribonuclease (BS-RNase) on bone marrow cells in vitro and in vivo Previously the immunosuppressive effect of this enzyme on T and B blastic transformation lymphocytes in bovine, pig and human (STANEK et al., 19781982-SOUCEK et al., 1981, 1986, 1996) without any harmful effect on NK cells was reported.Our former experiments showed that BS RNase inhibited regional GVH reaction, caused significant Prolongation of a skin graft survival and enhanced formation of GM-CFC colonies in mice and man (SOUCEK et al., 1983(SOUCEK et al., , 1986)).As new experiments, the binding of BS-RNase to normal bone marrow cells of pigs and the effect of this enzyme on the survival of pig's bone marrow cells ineubated 5 days in a refrigerator were investigated.Both experiments proved the ability of pig bone marrow cells to absorbed this enzyme and the positive effect of BS-RNase on the viability of these cells during 5 days storage in 4°C.In contrast to previous experiments in human (SOUCEK et al., 1986(SOUCEK et al., , 1996) ) and mice (SOUCEK et al., 1983), the colony-forming activity in pig bone marrow cell culture was not enhanced in the presence of BS RNase.Our irradiation and transplantation protocols used were inspired by the work of VAIMAN et al. (1981) studying skin and kidney grafts after partial lateral and conventional total lymphoid irradiation and bone marrow cell grafting.Our results although obtained with a limited number of bone marrow transplanted animals indicated a certain favourable effect of BS-RNase on the acceptance of donor bone marrow cells.Two reeipients from 7 ones accepted donor cells while no animal from 8 reeipients received donor's bone marrow cells pre-treated with BSA instead of BS-RNase.In above mentioned successful reeipients the detectable chimerism in leueocyte and blood group antigens was obtained and the pig 4 survived more than two years.The surviving of reeipients was probably also dependent on the lymphocyte and platelet levels after the irradiation and transplantation.The lowest number of lymphocytes and erythrocytes was observed from the forth day after the irradiation.Anaemia (mainly leukopenia) and probably trombocytopenia continued on individual dependence up to the death or up to the 17 days in the reeipient no 4 transplanted by the donor's cells ineubated by BS-RNase.The similar restoration of lymphocytes in partial lateral irradiation of pigs was published (VAIMAN et al., 1981;SAKAMOTO et al., 1988).Experiments with SLA, SLB and SLC compatible donor -reeipient pairs were performed only on three reeipients.However, in all these three cases the animals died between 12 and 17 days after the transplantation.Probably there was a difference in histocompatibility antigens which were not tested by our antisera because in the animal no 3 the GVH-like reaction was possible to recognise from the skin and organ rashes.Of course, the other transplantation antigens could play a role in this unexpected phenomenon.TRITTHART 471 Seiten, 119 Abbildungen, Tabellen, Springer Verlag, Wien -New York, 2000, ISBN 3-211-83046-4, DM 140,00;öS 980,00;sFr 127,50 Im Rahmen der Buchreihe "Ersatz-und Ergänzungsmethoden zu Tierversuchen", die von SCHÖFFL, SPIELMANN und TRITTHART herausgegeben wird, liegt inzwischen der 6. Band vor.Er enthält die Vorträge und Poster der 6., 7. und 8. internationalen Kongresse über Ersatz-und Ergänzungsmethoden zu Tierversuchen (TV), die innerhalb eines Jahres in Linz/Österreich stattfanden.Tierschutz (TS) ist gezielte Hilfe für das Tier und TS-Gesetze dienen dieser Hilfe, indem sie das Leben und das Wohlbefinden der Tiere schützen.Es ist ein erklärtes Ziel Tierversuche einzuschränken.Sie sind trotz erforderlicher Genehmigungsverfahren umstritten, zumal bei der Interpretation dessen, was als ethisch vertretbar angesehen wird, sich ein weites Feld unterschiedlicher Betrachtungsmöglichkeiten bietet.Im Gegensatz zu Vertretern, die eine generelle Abschaffung von Tierversuchen fordern, ist die erfolgreiche Suche nach Ersatz-und Ergänzungsmethoden für Tierversuche das verdienstvolle Anliegen dieses und der bereits vorliegenden Tagungsberichte.Ermöglicht er es doch dem Leser sich umfassend über diesbezügliche internationale Aktivitäten und wissenschaftliche Fortschritte im sensiblen Bereich der Entwicklung solcher Methoden zu informieren.Er trägt aber auch wesentlich zum objektiven Verständnis dieser häufig emotional diskutierten Thematik bei.Dieses Buch macht aber auch deutlich, dass bei überaus wünschenswerter Einschränkung bzw.bei Ersatz-und Ergänzungsmethoden in den verschiedenen Anwendungsbereichen, vor allem bei der biomedizinischen Forschung, die Ersetzbarkeit oder Kombination mit anderen Methoden möglich ist, immer aber mit zumindest gleichwertigen bzw.besseren Ergebnissen verbunden sein muss.Thematische Vielfalt kennzeichnet den vorliegenden 6.

Fig. 1 :
Fig. 1: Indirect immunofluorescence of bone marrow cells of miniature pig after I h incubation of cells with BS-RNase at 24° C a) Strong membrane fluorescence was seen when rabbit IgG against BS-RNase was used. 1 lOOx b) Control -normal rabbit IgG instead of rabbit IgG against BS-RNase was used. 1 lOOx