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Mouse thymic stromal cells induced functional maturation of medullary thymocytes
396
Hemopoiesis
25 June 1997 - Poster pmentahons
and 26.1 f 3.2 respectively) there were significant dferences in morphological composition of colonies: 2.75 f 0.5 were macrophage, 6.5 f 0.99 -granulocy& and 2.37 f 0.49 - mixed. Conduslon: It is suggested that G-CSF is capable to change the differetiation of hematopoietfc precursors in vitro, resulting in greater amount of granulocytic cell series without any influence on the whole hematopoietfc precursor pool.
1P.2.06.10 1 ;;mI
ontogeny of lymphocytes in miniature
M. Sinkora, J. Sinkora, Z. Rehakova, I. Splfchal, I. Trsbichavsky. Dept. lmmunol. and Gnotobioi., Inst. Micfobiol., Czech Academy of Sci., 549 22 Nov)i Htidek, The Czech Republic Introductfon: We studied the phenotypical development of lymphocyte subpopulations from the liver, thymus, umbilical blood, spleen, bone marrow, and intestine during prenatal ontogeny in miniature pigs by double- or Mple-colour flow cytometry (FCM). MaterlaIr and Methods: Freshly prepared suspensions of fetal cells from umbilical blood and organs were treated by hypotonic lysis (for depletion of erythrocytes). The surface staining with a panel of monoclonal antibodies directed against porcine leukocyte surface antigens (antiCD45, antiSWC3, LIG4, anti-CDP, anti-CDS&, an&CDS)&, anti-CD4. anti-CDS, antiCD8) was detected by isotype-specific secondary polyclonal antibodies conjugated to fluorescein isothiocyanate or R-phycoerythrtn. For triple-colour FCM, a biotinylated mAb was detected by a stmptavidin-Cy-Chmme complex. Results: First lymphoid cells with typical scatter characteristics and phenotype (SWC3-CD45+) can be found in the mid of morphogenesis (3Owday of gestation, DG 30) in the liver. However, these early lymphoid cells do not express either CD3E or slg. The first significant numbers of cells bearing CD3s on the surface were found in the thymus as early as thymocytes could be isolated for FCM (DG 40). Further analysis showed that most of them am TCR y/6+ T cells whereas TCR a/B+ T cells fom-redonly a minor population and that there is a significant proportion of CD4+CD8+ cells in the thymus that do not express CD3-8. First T cells in the spleen and umbilical blood were detected at about the DG 45, most of them being TCRa@+ whereas TCRyI6+ T cells footed only a minor population in this developmental stage. B cells appeared in the umbilical blood at the same time as T cells whereas in the spleen slgM+ cells were not observed before the DG 61. In the fetal intestine, first 6 cells and CD3-&+ T cells occur together as early as on the DG 45 (more TCR@+ T cells then TCRyQ+ T cells found). Lymphocytes formed only a minor subset in the fetal liver and bone marrow and their numbers slowly increased in bone marrow during ontogeny. By magnetic sorting we could, however, isolate quite a pure subsets of 6 and T lymphocytes from fetal liver as soon as on the DG 45. Conclurfonr: We identified the main differentiation stages and general differentiation trends during ontogeny of pig lymphocytes in the umbilical blood and fetal different organs. As lymphocytes and their subsets represent one of the important mechanisms of immunity, our phenotype analysis may help in understanding prenatal immunity and development of immune system in the mammals.
P.2.06.11
Induction of migration and apoptosis in bone marrow progenitors by low molecular weight collagen peptides
A.Yu. Yemelyanov, I.G. Kozlov, N.K. Gorlina, A.N. Chemdsev. Dept.of Immunofogy. Russian State Medica/ University,Moscow Russia
Introduction:As a consequence of degradation of collagen proteins during inflammation short collagen peptides am generated and am accumulated in the ama of inflammatory site. The peptides may defuse out of the inflammatory site and penetrate through tissue-blood banters due to their low molecular weight. As inflammation is characterized with deep changes of cell content in peripheral blood resultant from the hemopoietic rearrangements we studied the effects of cdlagen peptldes on migration, proliferation and regulation of apoptosis in bone marrow cells. Mate&Is and Methoda: Collagen peptides were prepared through 36 h hydrolysis of collagen type I with collagenase from Clostridium histokticum followed with ultrofiltratiin to obtain peptide fraction cl0 kDa, and were tested in the range of l-1000 &ml. Three dimensional (30) collagen matrix with collagen concentration of 1 m@mlwas prepared by adjusting the osmomolartty and DH of the collaoen tvos I solution in 0.05 M acetic acid to ohvsiolooical level: Bone marrow &IN were fractionated by panning and centrffu&ion~n a density gradient (1.066 dcm3, 1400 rpm). Cell migration in 3D collagen matrix was evaluated either by direct counting of migrating cells or, if the cells were prior labeled with 3H-thymidine, with B-counter. Apoptosis was assessed by a method of flow cytometry with hypotonic DNA staining with propidium iodide. Rewh Collagen pepttdes at concentrations 100-200 &ml magnified the number of bone marrow cells migrating in wllagen matrix. The activatoy effect
of the peptides was irreversible and developed by the first 8 h of cell incubation on collagen matrix. Activation of migration was observed only In population of low density bone marrow cells (XI ,066 s/cm3), which proliferated actively, were not glass adherent and did not express slg. Thus, coflagen peptfdes induced the capacity to migrate in collagen matrix in some ceils of precursor population. At the same concentrations collagen peptides induced apopto& in the fractions of low and high density cells. The effect reached maximum by 24 h of cell incubation with collagen peptides and was the most pronounced in the fraction of low density cells. Over the whole range of tested concentrations collagen peptides did not affect proliferation of bone marrow cells. Conclusion: We presume that low molecular weight collagen peptides are targeted on different bone marrow precursors, triggering the apopto&s in some cells and inducing the capacity of migration in others. Therefore, collagen peptides contribute to regulation of inflammation providing the selection of precursors for immigration out of bone marrow.
P.2.06.12
Mouse thymic stromal cells induced functional maturation of medullary thymocytes
Wei-Feng Chen, Li-Sheng Lu, Hai-Dong Dong, Qiang Wu. Dept. of Immunolog) Beding Medical U&et&y, Bejing, China
Introduction:After thymic selection, the newly emerged TCR+CD4+CDE-I CD4CDB+(SP) am non- or less functional, they undertake a functional maturation process in thymic medulla to acquire full immunocompetence. We have analyzed it the functional maturation is a spontaneous process or a process induced by interaction with medullary thymfc stromat cells. Materlals and Moth&x Both in vitro and in vivo experiments were performed. lsdated mouse Qa-2-CD4SP thymor@s were ‘cocultured with medullary-type thymic epithelial cell line MTECI in vitro. The Qa-2 expression was analyzed on FAGS and tympholdne production after Con A stimulation assessed. For apoptoeis detection, thymocytes were cocultured wfth mouse thymic dendrttic cell line (MTDC) and TUNEL+ cells counted. MTECI (H-e) cells wem injected into thymus (H-2b) to construct chimeric thymus mice. The spleen cells were assayed for the presence of H-2d restricted antigen specific T cells. Reeufb~ Mouse Qa-2CD4SP thymocytes after cocuftured with MTECI for 3 days, 85-89% Qa-2+ and produced the levels of 11-2,IL-4 and IL-6 virtually as high as those produced by freshly isolated Qa-2+CD4SP thymocytes. MTECl cells rescued thymocytes from apcptosis. By contrast, MTDC promoted apcp tosis of both CD4+CD8+ and CWSP thymocytes as revealed by percentage of hypodipkrid cells, DNA fragmentation and TUNEL+ cells. The spleen cells from chime& thymus mice exhibited significant levels of H-2d restricted antigen specific proliferation, IL-2 production and cytotoxfc kflling. These functions coukf not be detected in the spleen cells from control mice(H#) which wem injected intrathymlcally with saline. The intrathymic injected MTECI cells also induced remarkable tolerance to the H-2d antigen expressed on MTECI cells. Those the residence of MTECI cells within thymus could induce the development and maturation of H-2d restricted antigen specffic functional T cells. Wfth TUNEL staining, the clusters of cell apopto& were also shown in both cortex and medulla amas of normal mouse thymus sections. Conclusion:Mouse TCR’SP medullary thymocytes need interaction wfth in milieu thymic stroma for functional maturation, and at the same time to delete some (defect) cells. This process is pmceeded in thymic medufla and inferred as “Second Thymic Selection”. 1P.2.06.13 ) interaction between pre B cells and stromal cells depends on CD44 varlant isoformcl Lt. Giinthen ‘, R. Stauder 1,2,C. Schw&zter ‘, A. Bloem 3, M. van Drlel 3, E.-D. Kmuser4, F. SmadjaJoffe, S. Legras, A.G. Rolink’. lBaset/nstitute for Immunol~ Switzerland. * Uniwsity Hospital Innsbruck, Austria, 3Uniwsity Hospital Utrecht, The Nethedands, 4 University Hospitat Bertin, Getmaw 51NSERM, Wllejiuir.FMce The transmembrane surface molecule CD44 exists in multiple variant isoforms (CD44v) which am thought to be involved in hemopoiesis. speciffc migration of leukocytes and tumor cells. The expression of CD44 variant ccntaining isofomw at different stages of B cell development in mouse bone marrow was analyzed using specific mAbs. B22O+CD44vlO+cells were sorted and subsequent limiting dilution analysis on stmmal cells in the presence of IL-7 revealed the presence of a high frequency of clonabls pm B cells. In fact, this frequency was as high as that observed with sorted B220+ c-kit+ om B-l cells. Addition of the CD44vlO specific mAb as well as a fusion protein ~mprising mouse CD44v7-Inhuman IgG to cultures of pm 6 cells proliieratlng on stromal cefls in the presence of IL-7 led to an efficient and specific blocking of cell pmliieration. Thus, CD44vlO containing isofomw are expressed on early mouse pm B cell precursors and seem to play an important rote in the interaction between these precursors and the stromal cell compartment. To deffne the role of distinct CD44v regions in human myelorna prcgression
Hemopoiesis
25 June 1997 - Poster pmentahons
and 26.1 f 3.2 respectively) there were significant dferences in morphological composition of colonies: 2.75 f 0.5 were macrophage, 6.5 f 0.99 -granulocy& and 2.37 f 0.49 - mixed. Conduslon: It is suggested that G-CSF is capable to change the differetiation of hematopoietfc precursors in vitro, resulting in greater amount of granulocytic cell series without any influence on the whole hematopoietfc precursor pool.
1P.2.06.10 1 ;;mI
ontogeny of lymphocytes in miniature
M. Sinkora, J. Sinkora, Z. Rehakova, I. Splfchal, I. Trsbichavsky. Dept. lmmunol. and Gnotobioi., Inst. Micfobiol., Czech Academy of Sci., 549 22 Nov)i Htidek, The Czech Republic Introductfon: We studied the phenotypical development of lymphocyte subpopulations from the liver, thymus, umbilical blood, spleen, bone marrow, and intestine during prenatal ontogeny in miniature pigs by double- or Mple-colour flow cytometry (FCM). MaterlaIr and Methods: Freshly prepared suspensions of fetal cells from umbilical blood and organs were treated by hypotonic lysis (for depletion of erythrocytes). The surface staining with a panel of monoclonal antibodies directed against porcine leukocyte surface antigens (antiCD45, antiSWC3, LIG4, anti-CDP, anti-CDS&, an&CDS)&, anti-CD4. anti-CDS, antiCD8) was detected by isotype-specific secondary polyclonal antibodies conjugated to fluorescein isothiocyanate or R-phycoerythrtn. For triple-colour FCM, a biotinylated mAb was detected by a stmptavidin-Cy-Chmme complex. Results: First lymphoid cells with typical scatter characteristics and phenotype (SWC3-CD45+) can be found in the mid of morphogenesis (3Owday of gestation, DG 30) in the liver. However, these early lymphoid cells do not express either CD3E or slg. The first significant numbers of cells bearing CD3s on the surface were found in the thymus as early as thymocytes could be isolated for FCM (DG 40). Further analysis showed that most of them am TCR y/6+ T cells whereas TCR a/B+ T cells fom-redonly a minor population and that there is a significant proportion of CD4+CD8+ cells in the thymus that do not express CD3-8. First T cells in the spleen and umbilical blood were detected at about the DG 45, most of them being TCRa@+ whereas TCRyI6+ T cells footed only a minor population in this developmental stage. B cells appeared in the umbilical blood at the same time as T cells whereas in the spleen slgM+ cells were not observed before the DG 61. In the fetal intestine, first 6 cells and CD3-&+ T cells occur together as early as on the DG 45 (more TCR@+ T cells then TCRyQ+ T cells found). Lymphocytes formed only a minor subset in the fetal liver and bone marrow and their numbers slowly increased in bone marrow during ontogeny. By magnetic sorting we could, however, isolate quite a pure subsets of 6 and T lymphocytes from fetal liver as soon as on the DG 45. Conclurfonr: We identified the main differentiation stages and general differentiation trends during ontogeny of pig lymphocytes in the umbilical blood and fetal different organs. As lymphocytes and their subsets represent one of the important mechanisms of immunity, our phenotype analysis may help in understanding prenatal immunity and development of immune system in the mammals.
P.2.06.11
Induction of migration and apoptosis in bone marrow progenitors by low molecular weight collagen peptides
A.Yu. Yemelyanov, I.G. Kozlov, N.K. Gorlina, A.N. Chemdsev. Dept.of Immunofogy. Russian State Medica/ University,Moscow Russia
Introduction:As a consequence of degradation of collagen proteins during inflammation short collagen peptides am generated and am accumulated in the ama of inflammatory site. The peptides may defuse out of the inflammatory site and penetrate through tissue-blood banters due to their low molecular weight. As inflammation is characterized with deep changes of cell content in peripheral blood resultant from the hemopoietic rearrangements we studied the effects of cdlagen peptldes on migration, proliferation and regulation of apoptosis in bone marrow cells. Mate&Is and Methoda: Collagen peptides were prepared through 36 h hydrolysis of collagen type I with collagenase from Clostridium histokticum followed with ultrofiltratiin to obtain peptide fraction cl0 kDa, and were tested in the range of l-1000 &ml. Three dimensional (30) collagen matrix with collagen concentration of 1 m@mlwas prepared by adjusting the osmomolartty and DH of the collaoen tvos I solution in 0.05 M acetic acid to ohvsiolooical level: Bone marrow &IN were fractionated by panning and centrffu&ion~n a density gradient (1.066 dcm3, 1400 rpm). Cell migration in 3D collagen matrix was evaluated either by direct counting of migrating cells or, if the cells were prior labeled with 3H-thymidine, with B-counter. Apoptosis was assessed by a method of flow cytometry with hypotonic DNA staining with propidium iodide. Rewh Collagen pepttdes at concentrations 100-200 &ml magnified the number of bone marrow cells migrating in wllagen matrix. The activatoy effect
of the peptides was irreversible and developed by the first 8 h of cell incubation on collagen matrix. Activation of migration was observed only In population of low density bone marrow cells (XI ,066 s/cm3), which proliferated actively, were not glass adherent and did not express slg. Thus, coflagen peptfdes induced the capacity to migrate in collagen matrix in some ceils of precursor population. At the same concentrations collagen peptides induced apopto& in the fractions of low and high density cells. The effect reached maximum by 24 h of cell incubation with collagen peptides and was the most pronounced in the fraction of low density cells. Over the whole range of tested concentrations collagen peptides did not affect proliferation of bone marrow cells. Conclusion: We presume that low molecular weight collagen peptides are targeted on different bone marrow precursors, triggering the apopto&s in some cells and inducing the capacity of migration in others. Therefore, collagen peptides contribute to regulation of inflammation providing the selection of precursors for immigration out of bone marrow.
P.2.06.12
Mouse thymic stromal cells induced functional maturation of medullary thymocytes
Wei-Feng Chen, Li-Sheng Lu, Hai-Dong Dong, Qiang Wu. Dept. of Immunolog) Beding Medical U&et&y, Bejing, China
Introduction:After thymic selection, the newly emerged TCR+CD4+CDE-I CD4CDB+(SP) am non- or less functional, they undertake a functional maturation process in thymic medulla to acquire full immunocompetence. We have analyzed it the functional maturation is a spontaneous process or a process induced by interaction with medullary thymfc stromat cells. Materlals and Moth&x Both in vitro and in vivo experiments were performed. lsdated mouse Qa-2-CD4SP thymor@s were ‘cocultured with medullary-type thymic epithelial cell line MTECI in vitro. The Qa-2 expression was analyzed on FAGS and tympholdne production after Con A stimulation assessed. For apoptoeis detection, thymocytes were cocultured wfth mouse thymic dendrttic cell line (MTDC) and TUNEL+ cells counted. MTECI (H-e) cells wem injected into thymus (H-2b) to construct chimeric thymus mice. The spleen cells were assayed for the presence of H-2d restricted antigen specific T cells. Reeufb~ Mouse Qa-2CD4SP thymocytes after cocuftured with MTECI for 3 days, 85-89% Qa-2+ and produced the levels of 11-2,IL-4 and IL-6 virtually as high as those produced by freshly isolated Qa-2+CD4SP thymocytes. MTECl cells rescued thymocytes from apcptosis. By contrast, MTDC promoted apcp tosis of both CD4+CD8+ and CWSP thymocytes as revealed by percentage of hypodipkrid cells, DNA fragmentation and TUNEL+ cells. The spleen cells from chime& thymus mice exhibited significant levels of H-2d restricted antigen specific proliferation, IL-2 production and cytotoxfc kflling. These functions coukf not be detected in the spleen cells from control mice(H#) which wem injected intrathymlcally with saline. The intrathymic injected MTECI cells also induced remarkable tolerance to the H-2d antigen expressed on MTECI cells. Those the residence of MTECI cells within thymus could induce the development and maturation of H-2d restricted antigen specffic functional T cells. Wfth TUNEL staining, the clusters of cell apopto& were also shown in both cortex and medulla amas of normal mouse thymus sections. Conclusion:Mouse TCR’SP medullary thymocytes need interaction wfth in milieu thymic stroma for functional maturation, and at the same time to delete some (defect) cells. This process is pmceeded in thymic medufla and inferred as “Second Thymic Selection”. 1P.2.06.13 ) interaction between pre B cells and stromal cells depends on CD44 varlant isoformcl Lt. Giinthen ‘, R. Stauder 1,2,C. Schw&zter ‘, A. Bloem 3, M. van Drlel 3, E.-D. Kmuser4, F. SmadjaJoffe, S. Legras, A.G. Rolink’. lBaset/nstitute for Immunol~ Switzerland. * Uniwsity Hospital Innsbruck, Austria, 3Uniwsity Hospital Utrecht, The Nethedands, 4 University Hospitat Bertin, Getmaw 51NSERM, Wllejiuir.FMce The transmembrane surface molecule CD44 exists in multiple variant isoforms (CD44v) which am thought to be involved in hemopoiesis. speciffc migration of leukocytes and tumor cells. The expression of CD44 variant ccntaining isofomw at different stages of B cell development in mouse bone marrow was analyzed using specific mAbs. B22O+CD44vlO+cells were sorted and subsequent limiting dilution analysis on stmmal cells in the presence of IL-7 revealed the presence of a high frequency of clonabls pm B cells. In fact, this frequency was as high as that observed with sorted B220+ c-kit+ om B-l cells. Addition of the CD44vlO specific mAb as well as a fusion protein ~mprising mouse CD44v7-Inhuman IgG to cultures of pm 6 cells proliieratlng on stromal cefls in the presence of IL-7 led to an efficient and specific blocking of cell pmliieration. Thus, CD44vlO containing isofomw are expressed on early mouse pm B cell precursors and seem to play an important rote in the interaction between these precursors and the stromal cell compartment. To deffne the role of distinct CD44v regions in human myelorna prcgression