《【病毒外文文獻(xiàn)】1997 In vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero TGEV》由會(huì)員分享，可在線閱讀，更多相關(guān)《【病毒外文文獻(xiàn)】1997 In vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero TGEV（10頁珍藏版）》請(qǐng)?jiān)谘b配圖網(wǎng)上搜索。

INsTITuT PASTEURIELSEVIER Paris 1997 Res Immunol 1997 148 247 256 In vivo study of interferon alpha secreting cells in pig foetallymphohaematopoietic organs following in utero TGEV coronavirus injection I Splfchal I z ReMkova I M Sinkora 1 J Sinkora I I Trebichavsky I H Laude 2 and B Charley 2 IJ Division ofImmunology and Gnotobiology Institute ofMicrobiology ofthe Academy ofSciences ofthe Czech Republic 54922 Novy Hnidek Czech Republic and 2 Laboratoire de Virologie et Immunologie moleculaires INRA 78350 Jouy en Josas France SUMMARY Non infectious UV inactivated transmissible gastroenteritis virus TGEV was previ ously shown to induce interferon alpha IFNa secretion following in vitro incubation with blood mononuclear cells In this study pig foetuses at different stages of gestation were injected in utero with a partially UV inactivated wild TGEV or b fUlly UV inacti vated wild or dm49 4 mutant TGEV coronavirus Nucleated cells from foetal liver bone marrow spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFNa secretion by EUSPOT and ELISA techniques The administration of TGEV induced IFNa secreting cells in foetal Iymphohaematopoietic organs at mid gestation In contrast IFNa was not detected in control sham operated foetuses A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49 4 was associated with lower or absent IFNa in utero inducibility by mutant virus as compared with wild virus Row cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV or between dm49 4 and wild virus treated foetus cells with the exception of a reduction in percentages of polymorphonuclear cells in TGEV treated Iymphohaematopoietic tissues which is prob ably due to IFNa secretion The present data provided in vivo evidence of IFNa secretion at the cell level in foetallymphohaematopoietic organs Such IFNa secreting cells in Iym phohaematopoietic tissues may be the source of IFNa detected during foetal infections Key words Coronavirus Transmissible gastroenteritis virus IFNa ELISA ELISPOT Foetus Pig INTRODUCTION Interferon a IFNa is a critical component of early host non specific immune defence against viral infections It acts as both an antiviral agent and an immunomodulator as well as a cell growth inhibitor Several leukocyte populations are able to secrete IFNa in response to virus stimuli depending on the virus used and on whether viral infection of leukocytes is necessary or not Submitted January 9 1997 accepted Apri124 1997 Correspond ing author present address National Institute of Animal Health Kannondai Tsukuba Ibaraki 305 Japan 248 I SPLicHAL ET AL Monocytes are most often associated with pro duction of IFNa in response to infectious viruses Roberts et al 1979 Saksela et al 1984 whereas a distinct population of leukocytes among peripheral blood mononuclear cells PBMC referred to as natural interferon pro ducing cells NIPCs is able to secrete IFNa following exposure to non infectious viral struc tures Lebon et al 1982a Capobianchi et al 1985 Charley and Laude 1988 Human and porcine NIPCs have been characterized as highly infrequent non phagocytic non adherent non B non T MHC II and CD4 cells Cederblad and AIm 1990 Charley and Lavenant 1990 Sand berg et al 1990 Fitzgerald Bocarsly 1993 Nowacki and Charley 1993 Viral glycoproteins were suggested as being responsible for triggering synthesis of IFNa in NIPC Monoclonal antibodies mAbs to viral glycoproteins of herpes simplex virus type I transmissible gastroenteritis virus TGEV and Aujeszky s disease virus were shown to block virus induced IFNa secretion Lebon 1985 Charley and Laude 1988 Artursson 1993 However the precise nature of NIPCs as well as of the interactions between NIPCs and virus lead ing to IFNa production remains to be elucidated TGEV is an enteric coronavirus which causes acute and fatal diarrhoea as well as intense and early IFNa production in newborn piglets La Bonnardiere and Laude 1981 In vitro studies on IFNa induction by TGEV have shown that IFNa secreting cells IFNa SCs were detected among non adherent porcine PBMCs after expo sure to UV inactivated TGEV or glutaraldehyde fixed TGEV infected cells Charley and Laven ant 1990 Nowacki and Charley 1993 A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M in dm49 4 mutant TGEV was shown to be asso ciated with a defect in in vitro induction of IFNa Laude et al 1992 The epitheliochorial nature of placentation in pig prevents transfer of immunoglobulins or anti gens from mother to foetus which I rec1udes any immune activation of foetuses Sterzl et al 1966 This type of placentation together with multiparity relatively long term of gestation and size of foetuses makes this species suitable for studies on the development of the immune system In an earlier study on the prenatal ontog eny of porcine IFNa SCs we detected in vitro inducible IFNa SCs in pig foetal liver and other 1ymphohaematopoietic organs at very early stages of gestation Splichal et aI 1994 The present study was undertaken to evaluate in utero viral induction of IFNa SC in pig foetuses by TGEV either partially or fully UV inactivated at different stages of gestation We found that 10 or 20 h after in utero injection of TGEV in the umbilical cord of pig foetuses IFNa SCs were detected ex vivo in Iymphohaematopoietic tissues by the ELISPOT assay MATERIALS AND METHODS Animals Healthy pregnantgilts of miniature pig bred in the Laboratory of gnotobiology in Novy Hnidek were used in our experiments They had free access to water but were starved 12 hours before first surgery The gilts were subcutaneously s c premedicated with 1 mg of atropin sulphate Hoechst Biotika Slo vakia per 25 kg of body weight and they were anaesthetized with 1 5 2 5 of halothane Leeiva Czech Republic mixed with 0 and Np HCO 0 500 U Leciva Czech Republic and acetate FCM FCS FSC HCG IFN IFNa SC mAb MOC NIPC now cytometry analysis foetal calf serum forward scatter human chorionic gonadotropin interferon IFNa secreting cell monoclonal antibody mononuclear cell natural interferon producing cell PBL PBMC PBS PFU PMN SSC SWC3 TGEV peripheral blood leukocyte peripheral blood mononuclear cell phosphate buffered saline plaque fonning unit polymorphonuclear cell side scatter an antigen common for myelomonocyllc lineages transmissible gastroenteritis vrrus IN UTERO INDUCTION OF IFNa SECRETING CELLS 249 medroxyprogesterone 50 mg per 25 kg of body weight Upjohn Netherlands were intramuscularly i m injected In the first series of experiments four teen foetuses of52 82 and 101 days ofgestation were injected with partially UV inactivated TGEV 500 PRJ ml in 50 300 and 500 III of saline respectively via the umbilical vein when the umbilical cord was exteriorized after laparotomy and uterotomy of the gilts Control foetuses were subjected to the same sur gery but treated with equivalent volumes of the saline only The uterine and abdominal walls were sutured and gilts were placed in a postsurgical care unit They had free access to water but a limited amount of food Animals were treated with 1 500 000 U penicillin G Spofa Czech Republic s c and 0 5 g streptomycin Medexport Russia i m per 25 kg of body weight In the second series of experiments twenty one foetuses at 75 77 91 and 105 days ofgestation were injected via the umbilical vein 300 1 500 Ill with a fully UV inactivated wild or dm49 4 mutant TGEV initial titre of 6x 10 7 PFU per mI before inactiva tion The injected volume was proportionally adjusted to expected body weight The treatment of animals was the same as described above Cell suspensions The second uterotomy was performed 20 or 10 hours later in the first or second series of experi ments respectively The sows were anaesthetized foetuses were bled via umbilical cord arteries and blood samples containing 20 U of heparin ml of blood Leeiva Czech Republic were collected Cell suspensions from liver spleen both femurs and ster num were prepared by cutting these organs with scis sors in cold PBS Supernatants containing cells were collected after 10 min sedimentation at I g to remove debris and clumps PBMCs were prepared by centri fugation of diluted blood on Ficoll density gradient Pharmacia Sweden and red cells were depleted by hypotonic lysis with deionized water as described Splfchal et al 1994 In the case of a non adherent cell fraction isolation cells were incubated for 90 min in RPMI I640 with 20 foetal calf serum FCS Seromed Germany L glutamine 2 roM penicillin 100 UlmI and streptomycin 100 ug ml Gibco UK in 5 CO atmosphere at 37 C to remove plas tic adherent celts The number of nucleated cells and cell viability were calculated before assays IFNa assays Cell suspensions 200 Ill in RPMI with 10 FCS and antibiotics were log diluted in 3 6 wells of 96 well cell culture microplate Costar UK ELlS POT was performed as described Nowacki and Charley 1993 Cells in ELlSPOT plates or in tissue culture microplates were incubated in 5 CO atmosphere for 16 and 18 h respectively IFNa SC frequency was estimated by an ELISPOT assay using peroxidase labelled anti pig IFNa mAb F17 Spots were counted under binocular microscopy and the frequency was calculated from the total number of living nucleated cells and number of spots IFNa titres in supernatants from tissue culture microplates incubated for 18 h and in plasma were estimated by pig IFNa specific ELISA using the same antibodies as in the case of ELISPOT De Arce et al 1992 Nowacki and Charley 1993 Results arc expressed as IFNa unit m Production of IFNaJlFNa SC yield was estimated from the IFNa titre and the number of IFNa specific spots Flow cytometry analysis The following mouse mAbs directed against por cine leukocyte surface markers were used K252 1E4 anti CD45 74 22 15 anti SWC3 an antigen com mon for myelomonocytic lineages 1O 2H2 anti CD4 and MSA3 anti SLA DR Erythrocytes were removed from cell suspensions by hypotonic lysis of the cell pellet with water Leukocytes were washed and stained as described recently Cukrowska et aI 1996 briefly cells were treated with a primary mouse mAb and then with fluorescein conjugated swine anti mouse Ig polyclonal antibodies Sevac Czech Republic for double staining biotinylated mAbs were revealed by streptavidin phycoerythrin conjugate Immunotech France Cells were divided into two major populations on the basis of their size and internal complexity polymorphonuclear cells PMN with higher SSC parameter gated separately from mononuclear leukocytes MOC with lower internal complexity Flow cytometry data were obtained using a FACSort flow cytometer Becton Dickinson CA Propidium iodide was added to cells just before cytometry to prevent counting ofdead and damaged cells and at least 10 000 events were col lected Data were analysed using PC LYSYS 1 0 software Becton Dickinson CAl Virus High passage Purdue 115 wild strain of TGEV and dm49 4 mutant TGEV Laude et al 1992 were used as virus sources Procedures for virus preparation have been described previously Char ley and Laude 1988 Viruses were inactivated by UV irradiation In the first series of experiments TGEV was partially UV irradiated to obtain a resid ual infectivity titre of 5x 10 4 PFUlml In the second series of experiments both wild and dm 49 4 mutant virus at initial titres of 6x 10 7 PFU ml were fully inactivated before injection 250 I SPLiCHAL ET AL RESULTS Induction of IFNa SC following in utero injection of partially inactivated wild TGEV The optimal amount of partially inactivated wild TGEV allowing foetus survival after intrave nous injection at 54 days of gestation was deter mined in preliminary experiments Foetuses were thereafter infected with this amount of partially inactivated TGEV and other foetuses were sham operated as controls At 52 days of gestation only three foetuses two infected and one control sur vived till the next day dead foetuses infected by virus did not exhibit any macroscopic pathologi cal lesions when compared with the dead control foetus Only cell suspensions and plasma of sur viving foetuses were used for IFNa determina tions IFNa SCs or IFNa were detected in liver and bone marrow cells or plasma in only one of two TGEV treated foetuses already at mid gesta tion day 52 table I In the spleen they were found at later stages of gestation The highest IFNa SC frequency was observed in foetal liver A slightly higher IFNa SC frequency was detected in non adherent cells compared with that in total cells table I 101 days The absence of lFNa SCs in spleen cells at 52 days may be due to the limited number of cells The highest IFNa SC frequency was observed by 101 days of gestation whilst IFNa yield production of IFNa per cell was roughly constant table II No IFNa secretion in bone marrow cell culture supernatants could be detected at 52 days ofgestation table II although low numbers of IFNa specific spots were detected in one stimulated foetus table I Table I IFNa secreting cells in pig foetal Iymphohaematopoietic organs and plasma IFNa levels 20 h after experimental in utero injection of partially UV inactivated wild TGEV coronavirus IFNa SCfr uency IFNa level spots per 1 cells units per ml Day of gestation Liver Spleen Bone marrow Plasma 52nd n 2 26 0 o 2 1 0 4800 0 82nd n 4 84 5 57 7 2 2 1 1 14 8 13 5 9200 2870 IOlst n 3 47 3 23 4 6 3 0 2 7 7 4 7 10 190 2 920 IOlst a n 3 66 3 23 7 ND ND Results are expressed as individual data or as means standard deviation Day of gestation day of stimulation by TGEV a non adherent fraction b low number of cells ND non detected Numbers of sham operated controls on S2nd 82nd and JOist day of gestation were 1 2 and 2 respectively No IFNa SC or IFNa titres were detected in Iymphohaematopoietic organs or plasma of controls Table II IFNa yield per cell in pig foetallymphohaematopoietic cell cultures 20 h after experimental in utero injection of partially UV inactivated wild TGEV coronavirus Day of gestation 52 nd n 2 82nd n 4 l Olst n 3 1OIst a n 3 Liver 0 7 0 1 9 0 8 0 3 0 2 0 2 0 IFNa secretion units per IFNa SC Spleen O b 0 3 0 1 0 2 0 1 ND Bone marrow o 0 9 0 8 0 2 0 1 ND aJ Non adherent fraction b low number of cells ND non detected No IFNa titres were detected in cell culture supernatants of Iymphohaematopoietic organs of controls IN UTERO INDUCTION OF IFNa SECRETING CELLS 251 As controls IFNa titres were determined in the plasma of TGEV injected foetuses High IFNa titres were found in foetal plasmas at 82 and 101 days of gestation table I Only one TGEV injected foetus had plasma IFNa at 52 days of gestation No lFNa specific spots or IFNa secretion was found in control sham operated foetuses which were subjected to in utero injection of saline only data not shown Flow cytometry FCM analysis was per formed in 52 and IOI day old foetuses SSCIFSC dot plot analysis showed a lower per centage of PMN cells in virus injected foetal pig organs fig 1 FCM of leukocyte cell markers did not show significant differences between con trol and TGEV injected foetuses data not shown After evaluation of the results from this first series of experiments we reduced the length ofin utero stimulation from 20 to 10 hours in order to analyse IFNa secretion at its expected time of maximal production Induction of IFNa SC following in utero injection offully inactivated wild and dm49 4 mutant TGEV In a second series of experiments using fully inactivated virus differences in IFNa induction in pig foetuses stimulated by wild or dm49 4 mutant TGEV were analysed In order to exclude any possible IFNa secretion by cells of monocytic macrophage lineage only non adherent cells were used as performed with liver cells at 101 days table I The main dif ferences observed between the two series of experiments were a much lower plasma IFNa level a reduced IFNa SC frequency in liver and spleen and the absence of IFNa SCs in foe tal bone marrow table III compared with table I IFNa yields per cell were similar tables II and IV Neither plasma IFNa nor IFNa SCs in organs were found in dm49 4 injected foetuses with the exception of very low IFNa SC numbers in liver and spleen of 75 day old foetuses tables III and IV Flow cytometry showed lower percentages of PMN cells in liver and bone marrow of wild TGEV infected foetuses than in dm49 4 treated foetuses with one exception the liver of 105 day old foetuses fig 2 No significant changes in leukocyte marker expression were observed data not shown DISCUSSION Intraamniotic infections provoke abortion infertility foetal death and abnormal foetal devel opment and are associated with increased levels of amniotic inflammatory cytokines Gravett et al 1994 Romero et al 1994 Dudley et aI 1996 J FN was found not only in human foetuses during intraamniotic rubella infection Lebon et al 1985 but also in the amniotic fluid of preg nant women without clinical signs of congenital virus infection Lebon et al 1982b Following our previous demonstration that in vitro inducible IFNa SCs were present at early stages of gesta tion in porcine foetal lymphohaematopoietic organs SpHchal et al 1994 the aim of the present study was therefore to evaluate at the cell level the secretion of IFNa following in utero intravenous injection of TGEV in pig foetuses at different stages of gestation The study was divided into two parts a induction of IFNa secretion by partially inactivated wild TGEV for which IFNa secretion was analysed in total cell populations first series of experiments and b induction of IFNa secretion in the non adherent IFNa SC fraction presumably NIPC like cells after exposure to non infectious TGEV Wild and dm49 4 mutant TGEVs were compared in order to evaluate the influence of an amino acid point mutation in protein M on the IFNa inducing properties in vivo second series of experiments The experimental approach using open sur gery is an efficient procedure for in vivo infection of foetuses via the umbilical cord vein It has however several disadvantages such as loss of amniotic fluid damage of foetal membranes and possible damage of the umbilical cord Kovaru et al 1971 In the present study we observed high mortality of pig foetuses at 52 days of gestation 252 I SPLiCHAL ET AL o 1000 Stimulated fetus o O r o o o llCl A 0 c a 0 C l 0 I C l lI Io r r r J r Non stimuiated fetus a o g 7 P orC lnt of PMN CIUS 1 7 0 c 50 n 10 umbl c 1 blood bone m ilrTC liver umbll cal blood bone marro r 5 lnd d3y of g st toon o NO STlMU TEO 0 STIMUl ATEO BY LNE TGEV 01 SI day f o st al on Fig 1 FCM of umbilical cord blood bone marrow and liver cells of 52 and IOl day old foetuses after in utero injection of control medium or TGEV Dot plot analysis of foetal bone marrow cells 101 days of gestation from control a or TGEV injected b foetuses Percentages of PMN cells in the different cell suspensions at 52 and 101 days of gestation c Number of foetuses as indicated in table 1 even in the case of one control foetus treated by saline only which could be due to easier damage of the umbilical cord at this period of gestation than with older foetuses The main finding of the present study was that IFNa SCs were found as early as 52 days of ges tation in foetal liver and bone marrow following in utero injection of partially UV inactivated IN UTERO INDUCTION OF IFNa SECRETING CELLS 253 Table Ill IFNa secreting cells in pig foetallymphohaematopoietic organs and plasma IFNa levels to h after experimental in utero injection of UV inactivated wild and dm49 4 mutant TGEV coronavirus Liver Wild virus Mutant virus Day of gestation wild mutant IFNa SCfre uency spots per 10 cells Spleen Wild virus Mutant virus IFNalevels units per ml Plasma Wild virus Mutant virus 75th n 3 3 77th n 211 91st n 3 3 105th n 3 3 3 4 2 9 0 7 1 7 3 7 1 4 0 1 0 1 0 1 0 1 o o o 1 9 1 4 0 2 4 28 8 ll 5 18 1 1 7 0 1 0 1 o o o 990 120 ND 1480 1180 1400 780 o o o o IFNa ELISPOT assay was performed with non adherent cells