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Functional consequences of contact interaction between lymphocytes and fibroblasts in vitro
Hemopoiesis
25 June 1997 - Poster presentations Materiels and Methods: Human spleens were obtained from multi-organ donors. Flbmblast-like stromal cells were obtained by culturing spleen cell suspension at high density for l-2 weeks and subsequent expansion of adherent cells. B lymphocytes were purified by a combination of SRBCAET resetting and magnetic beads separation then they were stimulated wkh SAC particles for 2 days. The obtained blasts were co-cultured with stromal cell monolayers. The cultures were examined for B cell recovery, immunoglobulin and cytokine production. Rwu~ In vitro activated splenic lymphocytes secrete up to 10 times more IgG when cultured over monolayers of stromal cells. Stmmal cells activity involved both support of B cell survival and differentiation of B cells into plasma cells as assessed by morphology and CD38 expression. Addition of neutralizing antibodies aaainst IL-6 and TNFa abolished increased IaG secretion. Addition of recombin& IL-6 and TNFa to B cells cultured alone mimicked the effect of stmmal cells althouah to a smaller extent. This suggests possible importance of __ B cell-stmmal cell adhesion. Conduelone: Fibroblasts-like stromal cells derived from human spleen support terminal differentiation of B lymphocytes
Abnormal stromal microenvironment8 malntain chronic inflammation A.N. Akbar, H. Hyde, N.J. Botthwick, D. Pilling, D. Scheel-ToelIner, M. Salmon. Dept. of Clinical Immunology, Royal Free Hospital and Dept of Rheumatology; Birmingham, Introductton: The majority of activated T cells which are generated in an immune response are cleared, after disease resolution, by apoptosis. This is important for the maintainence of homeostasis. However, mechanisms which prevent the apoptosis of a proportion of the activated T cells are important for the retention of a primed population, which may take part in secondary responses. Methods:IL-2 deprived T cell lines, which were a model for effector cells generated in viw, were deprived of this cytokine which results in apoptosis. These cells were cultured with monolayers of human embryonic lung fibroblasts which can prevent apoptosis. Reeutts: Co-culture of IL-2deprived T cells with fibroblasts prevented apoptosis but did not induce proliferation. The fibmblasts induced Bcl-XL but not Bcl-2 expression in the rescued T cells. Furthermore, the fibroblast factor, which was a secreted product, induced the upregulation of the antioxidant mdecule glutathione in the rescued T cells, which was directly related to the increased survival. Conduslon: As the balance between death and survival is important for leucocyte homeostasis, it would be predicted that the excessive retention of primed T cells by abnormal stromal microenvironments may lead to chronic inftammation. We have demonstrated that this may be one reason for the chronic persistence of primed T cells in the joints of rheumatoid arthritis patients.
P.2.06.07
Functlonal consequences of contact interaction between lymphocytes _. _ and flbroblasts in vitro
LG. Kozlov, N.K. Godina, A.Yu. Yemelyanov, A.N. Cheredeev. Department of Immunol~, Russian State Medical UniversiM Moscow, Russia Introductfon: Migration of immunocompetent cell over lymphoid and non-lymphoid organs and tissues is on of the stages of recirculation and homing. During their migration cells of immune system are involved in contacts with tissue cells that, obviously, may change functional state of immunocompetent cells as well as of non-lymphold cells. In the present work we studied cell functional consequences resulted from in vitro contact between lymphocyte and fibroblast, which is the most widely distributed stromal cell. Materialsand Methods:The fraction of CBA mice splenocytes, deprived of erythrocytes and glass adherent cells, was taken as lymphocytes. Flow cvtometw analvsis showed that the solenocvte fraction contained 54.4 f 8.2% of T cells, 37.9-k 5.4% of B cells and’6-8% of 0 cells. Three lines of fibroblasts as mouse highly transformed L929, mouse minimally transformed 3T3 and human embrvonic fibroblasts Ml9 were emoloved. Lvmohocvtes were added to fibmblasts pior cultivated for 24 h (at a ratio’rangedfrom 1:4 to 1:l0). By various periods of co-cultlvatton we investigated the number of lymphocytes adhering to-fibroblasts. ftbmblast RNA synthesis, proliferation and secretion of collagen and non-collagen proteins. Resuttez By the first 2 h of lymphocyte-flbroblast co-cultivation over 20% of lymphocytes have adhered to fibrobtast confluent. In non-confluent fibmblast cultures within the first 12 h the contact interaction assured time-dependent decrease in RNA synthesis and inhibition of proliferation. The lymphocyte inhibitory effect was not associated neither with their cvtotoxicitv nor with cvtokines secreted by them. The degree of decrease in RNA and DNA synthesis depended on a lymphocyte-fibmblasts ratio and the level of Cbroblast transformation (with minimum oppression in L929 and maximum oppression in 119). In parallel with the inhibition of RNA and DNA synthesis a pronounced augmentation in secretton of collagen and non-collagen proteins was observed. When the time of
395
lymphocyte-fibmblast co-culturing was prolonged RNA synthesis in fibroblasts significantly grew and become equal (or sometimes exceeded control values) to that of control that was seen by 36 h of co-cultivation in L929 fibroblast, and by 72 h in 3T3 and Ml9 fibroblasts. Simultaneously with this, fibroblast proliferation increased, but remained lower then in control. Secretion of collagen and non-collagen proteins constantly exceeded that in control. In 72-120 h of lymphocyte-fibroblast co-cultivation RNA synthesis and proliferation repeatedly decreased and then remained lower then in control cultures even after a passage. Conduslon: Thereby, the oppression of proliferation (which may be associated with the inhibition of RNAsynthesis) and enhancement offibmblast specific function may be resultant from lymphocyte-fibroblast contact interaction.
mp.2.06.08
I
Phenotypic characterization of myelomonocytic haemopoietic ceriters In plg
’ c$s&~rimary
2. Rehakova’ , J. Sinkoral, M. Sinkoral, B. Cukrowska *, I. Spllchal’ . ‘Dept. Immunol. and &otobid., I&t. Microbial.; Acad. Sci. of the C&h Republic,’ Novy Hradek, Czech Republic, 2Dept. Immunol. and Gnotobiol., Inst. Microbial..,Acad. Sci. of the Czech Republic, Prague, Czech Republic Introduction: In our effort to look for possible producers of inflammatory cytokines in pig fetuses we studied the phenotype of non-lymphoid leukocytes and their precursors isolated from primary haemopoiettc centers (fetal liver and bone marrow) bv the method of double-colour flow cvtcmetrv (FCM). The phenotype of cyckng precursors was determined by a combination of surface marker staining followed by DNA quantification in ethanol-fixed cells. Materlals and Methods: Single cell suspensions from organs studied were used either intact or treated with an NH4CI based lysing solution or with hypotonic shock. Hypotonic lysis proved to be the best to enrich cell preparations for the CD45+ cells. We used a panel of monoclonal antibodies (moAbs) recognizing surface markers expressed by cells of the myelomonccytic lineage in the pig: antiCD45, antiCD14. anti-MHC II, antiSWC3. anti-SWCI, MU, Ml13 and MlL4. Subsets of live and undamaged leukocytes were analyzed with respect to light scatter characteristics and surface marker expression. For DNA staining in ethanol fixed cells the 7-AAD probe was used. Results: Fetal liver and bone marrow in pigs consist mainly of more or less mature cells of the myelomonocytic lineage. Lymphocytes are rare, especially when the liver is a dominant hemopoietic organ. We set gates to analyze mononuclear (MN) and polymorphonuclear (PMN) cells separately and we found typical differentiation stages residing also in adult bone marrow, although relative proportions of leukocyte subsets depended on the age of fetuses. Simultaneous surface marker and DNA staining allowed us to detect cycling precursors and to characterize their surface phenotype. Surprisingly, almost all surface antigens studied that were originally found on mature stages of leukocytes can also be found on cycling bone marrow and fetal liver precursors. Conclusions: We identified different subsets and differentiation stages of the myelomonccytic lineage in fetal pigs. Because fetal liver represents the most convenient source of leukocytes in early fetuses, our phenotype analysis will help to purify different hemopoietic precursors and leukocyte populations early in ontogeny by magnetic sorting or using the light scatter and/or immunofluorescence in FCM and to test their capability of producing inflammatory cytokines following stimulation in vitro.
P.2.06.09
Differentiation changes of hematopoietic precursors after In v/fro incubation of bone marrow cells from irradiated mice with granulocyte colony-stimulating factor
L.A. Cheredeeva, K.S. Chertkov. Department of Hematology, hstitute of Biophysics, Moscow, Russia Introduction: It is well known that growth factors in viw are able to increase the number of hematopoietic precursors. So, it was interesting to study, if such an effect can be seen after in vitro incubation of granulocyte colony-stimulating factor (G-CSF) with bone marmw cells from subkrthally irradiated mice. _ Materials and Methods: Mice (C57BL x CBA)Fl were used. Bone marrow cells (1 x @/ml) from sublethally irradiated mice (4 Gr) were incubated with G-CSF (2 &ml) during 3 h and then were injected into lethally irradiated syngeneic recipients. After 10 days the number of spleen colonies was determined. Reeu& No significant increase in the number of spleen colonies was shown after incubation of bone marrow cells with G-CSF. In spite of this, morphological anatvsis has revealed siantficant differences in control and experimental groups. In control (without incubation with G-CSF), the whole colony number was 16.6 f 1.49, while cluster number was 24.6 f 3.37. Among colonies analyzed 5.8 f 0.87 belonged to macrophage, 3.6 f 0.62 - to granulocytic, and 1.I2 f 025 - to mixed. On the other hand, in experimental group (incubation with G-CSF), despite of no differences in the numbers of colonies and clusters (11.6 f 1.62
25 June 1997 - Poster presentations Materiels and Methods: Human spleens were obtained from multi-organ donors. Flbmblast-like stromal cells were obtained by culturing spleen cell suspension at high density for l-2 weeks and subsequent expansion of adherent cells. B lymphocytes were purified by a combination of SRBCAET resetting and magnetic beads separation then they were stimulated wkh SAC particles for 2 days. The obtained blasts were co-cultured with stromal cell monolayers. The cultures were examined for B cell recovery, immunoglobulin and cytokine production. Rwu~ In vitro activated splenic lymphocytes secrete up to 10 times more IgG when cultured over monolayers of stromal cells. Stmmal cells activity involved both support of B cell survival and differentiation of B cells into plasma cells as assessed by morphology and CD38 expression. Addition of neutralizing antibodies aaainst IL-6 and TNFa abolished increased IaG secretion. Addition of recombin& IL-6 and TNFa to B cells cultured alone mimicked the effect of stmmal cells althouah to a smaller extent. This suggests possible importance of __ B cell-stmmal cell adhesion. Conduelone: Fibroblasts-like stromal cells derived from human spleen support terminal differentiation of B lymphocytes
Abnormal stromal microenvironment8 malntain chronic inflammation A.N. Akbar, H. Hyde, N.J. Botthwick, D. Pilling, D. Scheel-ToelIner, M. Salmon. Dept. of Clinical Immunology, Royal Free Hospital and Dept of Rheumatology; Birmingham, Introductton: The majority of activated T cells which are generated in an immune response are cleared, after disease resolution, by apoptosis. This is important for the maintainence of homeostasis. However, mechanisms which prevent the apoptosis of a proportion of the activated T cells are important for the retention of a primed population, which may take part in secondary responses. Methods:IL-2 deprived T cell lines, which were a model for effector cells generated in viw, were deprived of this cytokine which results in apoptosis. These cells were cultured with monolayers of human embryonic lung fibroblasts which can prevent apoptosis. Reeutts: Co-culture of IL-2deprived T cells with fibroblasts prevented apoptosis but did not induce proliferation. The fibmblasts induced Bcl-XL but not Bcl-2 expression in the rescued T cells. Furthermore, the fibroblast factor, which was a secreted product, induced the upregulation of the antioxidant mdecule glutathione in the rescued T cells, which was directly related to the increased survival. Conduslon: As the balance between death and survival is important for leucocyte homeostasis, it would be predicted that the excessive retention of primed T cells by abnormal stromal microenvironments may lead to chronic inftammation. We have demonstrated that this may be one reason for the chronic persistence of primed T cells in the joints of rheumatoid arthritis patients.
P.2.06.07
Functlonal consequences of contact interaction between lymphocytes _. _ and flbroblasts in vitro
LG. Kozlov, N.K. Godina, A.Yu. Yemelyanov, A.N. Cheredeev. Department of Immunol~, Russian State Medical UniversiM Moscow, Russia Introductfon: Migration of immunocompetent cell over lymphoid and non-lymphoid organs and tissues is on of the stages of recirculation and homing. During their migration cells of immune system are involved in contacts with tissue cells that, obviously, may change functional state of immunocompetent cells as well as of non-lymphold cells. In the present work we studied cell functional consequences resulted from in vitro contact between lymphocyte and fibroblast, which is the most widely distributed stromal cell. Materialsand Methods:The fraction of CBA mice splenocytes, deprived of erythrocytes and glass adherent cells, was taken as lymphocytes. Flow cvtometw analvsis showed that the solenocvte fraction contained 54.4 f 8.2% of T cells, 37.9-k 5.4% of B cells and’6-8% of 0 cells. Three lines of fibroblasts as mouse highly transformed L929, mouse minimally transformed 3T3 and human embrvonic fibroblasts Ml9 were emoloved. Lvmohocvtes were added to fibmblasts pior cultivated for 24 h (at a ratio’rangedfrom 1:4 to 1:l0). By various periods of co-cultlvatton we investigated the number of lymphocytes adhering to-fibroblasts. ftbmblast RNA synthesis, proliferation and secretion of collagen and non-collagen proteins. Resuttez By the first 2 h of lymphocyte-flbroblast co-cultivation over 20% of lymphocytes have adhered to fibrobtast confluent. In non-confluent fibmblast cultures within the first 12 h the contact interaction assured time-dependent decrease in RNA synthesis and inhibition of proliferation. The lymphocyte inhibitory effect was not associated neither with their cvtotoxicitv nor with cvtokines secreted by them. The degree of decrease in RNA and DNA synthesis depended on a lymphocyte-fibmblasts ratio and the level of Cbroblast transformation (with minimum oppression in L929 and maximum oppression in 119). In parallel with the inhibition of RNA and DNA synthesis a pronounced augmentation in secretton of collagen and non-collagen proteins was observed. When the time of
395
lymphocyte-fibmblast co-culturing was prolonged RNA synthesis in fibroblasts significantly grew and become equal (or sometimes exceeded control values) to that of control that was seen by 36 h of co-cultivation in L929 fibroblast, and by 72 h in 3T3 and Ml9 fibroblasts. Simultaneously with this, fibroblast proliferation increased, but remained lower then in control. Secretion of collagen and non-collagen proteins constantly exceeded that in control. In 72-120 h of lymphocyte-fibroblast co-cultivation RNA synthesis and proliferation repeatedly decreased and then remained lower then in control cultures even after a passage. Conduslon: Thereby, the oppression of proliferation (which may be associated with the inhibition of RNAsynthesis) and enhancement offibmblast specific function may be resultant from lymphocyte-fibroblast contact interaction.
mp.2.06.08
I
Phenotypic characterization of myelomonocytic haemopoietic ceriters In plg
’ c$s&~rimary
2. Rehakova’ , J. Sinkoral, M. Sinkoral, B. Cukrowska *, I. Spllchal’ . ‘Dept. Immunol. and &otobid., I&t. Microbial.; Acad. Sci. of the C&h Republic,’ Novy Hradek, Czech Republic, 2Dept. Immunol. and Gnotobiol., Inst. Microbial..,Acad. Sci. of the Czech Republic, Prague, Czech Republic Introduction: In our effort to look for possible producers of inflammatory cytokines in pig fetuses we studied the phenotype of non-lymphoid leukocytes and their precursors isolated from primary haemopoiettc centers (fetal liver and bone marrow) bv the method of double-colour flow cvtcmetrv (FCM). The phenotype of cyckng precursors was determined by a combination of surface marker staining followed by DNA quantification in ethanol-fixed cells. Materlals and Methods: Single cell suspensions from organs studied were used either intact or treated with an NH4CI based lysing solution or with hypotonic shock. Hypotonic lysis proved to be the best to enrich cell preparations for the CD45+ cells. We used a panel of monoclonal antibodies (moAbs) recognizing surface markers expressed by cells of the myelomonccytic lineage in the pig: antiCD45, antiCD14. anti-MHC II, antiSWC3. anti-SWCI, MU, Ml13 and MlL4. Subsets of live and undamaged leukocytes were analyzed with respect to light scatter characteristics and surface marker expression. For DNA staining in ethanol fixed cells the 7-AAD probe was used. Results: Fetal liver and bone marrow in pigs consist mainly of more or less mature cells of the myelomonocytic lineage. Lymphocytes are rare, especially when the liver is a dominant hemopoietic organ. We set gates to analyze mononuclear (MN) and polymorphonuclear (PMN) cells separately and we found typical differentiation stages residing also in adult bone marrow, although relative proportions of leukocyte subsets depended on the age of fetuses. Simultaneous surface marker and DNA staining allowed us to detect cycling precursors and to characterize their surface phenotype. Surprisingly, almost all surface antigens studied that were originally found on mature stages of leukocytes can also be found on cycling bone marrow and fetal liver precursors. Conclusions: We identified different subsets and differentiation stages of the myelomonccytic lineage in fetal pigs. Because fetal liver represents the most convenient source of leukocytes in early fetuses, our phenotype analysis will help to purify different hemopoietic precursors and leukocyte populations early in ontogeny by magnetic sorting or using the light scatter and/or immunofluorescence in FCM and to test their capability of producing inflammatory cytokines following stimulation in vitro.
P.2.06.09
Differentiation changes of hematopoietic precursors after In v/fro incubation of bone marrow cells from irradiated mice with granulocyte colony-stimulating factor
L.A. Cheredeeva, K.S. Chertkov. Department of Hematology, hstitute of Biophysics, Moscow, Russia Introduction: It is well known that growth factors in viw are able to increase the number of hematopoietic precursors. So, it was interesting to study, if such an effect can be seen after in vitro incubation of granulocyte colony-stimulating factor (G-CSF) with bone marmw cells from subkrthally irradiated mice. _ Materials and Methods: Mice (C57BL x CBA)Fl were used. Bone marrow cells (1 x @/ml) from sublethally irradiated mice (4 Gr) were incubated with G-CSF (2 &ml) during 3 h and then were injected into lethally irradiated syngeneic recipients. After 10 days the number of spleen colonies was determined. Reeu& No significant increase in the number of spleen colonies was shown after incubation of bone marrow cells with G-CSF. In spite of this, morphological anatvsis has revealed siantficant differences in control and experimental groups. In control (without incubation with G-CSF), the whole colony number was 16.6 f 1.49, while cluster number was 24.6 f 3.37. Among colonies analyzed 5.8 f 0.87 belonged to macrophage, 3.6 f 0.62 - to granulocytic, and 1.I2 f 025 - to mixed. On the other hand, in experimental group (incubation with G-CSF), despite of no differences in the numbers of colonies and clusters (11.6 f 1.62