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The participation of granulocytes in osteogenesis
120
23 June 1997 - Poster presentations
Phagocyte activation adfunction
P.3.08.23
Fosslbie sequence of events resuiting in resolution of infection with Francisella fdamnsis in mice
A. Macela ‘, M. Kroca I.*, L. Hemychova I, H. Kovamva ‘, J. Stulik ‘, 2. Krocova I. ’ Institute for Immunotog~ Pmkyne Military Medical Academ)! 500 01 Hmdec Kmlove, Czech Republic, 2Dept. Micmbiol., National LIefence Research Establishment, S-901 82 Umea, Sweden Introduction: Regulatory events dudng the immune response against intracellular bacterial pathogens are commonly understood. On the other hand, the inductive phase and decisive mechanisms operating in the course of primary response remain unclear. Our effort was focused on review of possible inductive interactions leading to the resolution of primary infection of mice with intracellular bacterial pathogen Ffancise//a tulafensls and resulting in ultimate protective immunity. Matertals and Methods: Subcutaneous infection of mice with Fran&e//a tularensis strain LVS in sublethal doses was used in this study. Several in vitro reactions of antibody with antigen, test of cytotoxicity of several populations of effector cells based on the release of 51chromium from target cells, and the levels of extracellular cytokines measured by commercial ELISA sets were utilized for screening of immunological status of host mice. ResuRs: Primary immune response against intracellular bacterial pathogen Fmncise//a Warensis can be induced by possible direct primary interactions of microbes with macrophages, NK cells, and B cells. These primary interactions lead to the production of several cytokines. Among them, the early production of IL-10 is probably responsible for increasing lytic activity of NK cells. Concomitant production of IFN-gamma is responsible for priming of macrophages for metabolic and functional activation. Simultaneously, with the production of TNF-alpha and IL-6 by macrophages, and the presence of specific antitularemic antibodies, the increase of number of bacteria in organs of infected mice was diminished. According to the cytokine profile (11-2,IL-4) Thl and Th2 branches of immunoregulatory mechanism are operating in this stage of infection. In the course of the highest activity of regulatory lymphocytes, cytotoxic macrophages and the effector cells using specific antibodies for extracellular FmnciseMatularensis killing occur. Together with the appearance of above-mentioned activities, the absolute decrease of microbes in organs of infected mice was evident. Conclusion: In summary, our results demonstrate that for resolution of primary Fmncisella tolamnsis infection of mice, the early production of IL-IO and TNF-alpha play most important role. The presence of antitularemic antibodies has dominant role in effective elimination of microbes from tissues of infected mice.
P.3.08.24
TNF-ar plus IFNq induce expression of siaioadhesin receptor by stromai bone marrow macrophages (M#)
S.A. Kusmartsev’, J. Navarro’, I. Angulo2, M. Danylets’, J.L. Subiza*. ‘Lab Siomodets of Tomsk Research Center Tomsk, Russia, * Servicio de lnmunologia Hospital Univemitario San Caries, Madrid, Spain Stromal murine Mg in adult bone marrow and lymphoid organs express a specific adhesion receptor which interact with sialited structures on developing myeloid cells and also on sheep erythrocytes. To test whether combination of TNF-n plus IFN-y, that is known as inductor differentiation of myeloid cells, could synergize for induction of sialoadhesin receptor, we used resetting assay with 51Cr-labeled sheep erythrocytes to detect functional surface expression of sialoadhesin on bone marrow derived M+. Thls method was combined with crystal violet assay and reverse centrlfugatfon. Our result showed that treatment of bone marrow M# by TNFa or IFN-y alone for 24 h, did not induce of erythrocyte binding to MI$ as compared with control. The highest binding of 51Cr-labeled erythmcytes observed after culturing of M+ in presence TNFa plus IFN-y (4 to 5-fold increase in 3 separate experfments). Addition of IL-1 to this combination did not enhance observed effect. Using IL-1 alone, as well as LPS or M-CSF had a weaker stimulation effect. As showed experiments with blocking anti-sialoadhesin monoclonal antibodies and also with neuramlnidasae-treated erythrocytes this TNF&lFN-y- inducing effect was mediated by siafoadhesln. We also found that treatment of M# with this cytokine combination for 24 h stimulated attaching of these cells to plastic substrata. Taken together our data demonstrate that TNF-a plus IFN-y has strong inducing effect on adhesion molecule expression by bone marrow M&. On the other hand, combination of TNFa and IFN-y is well known as inductor expression of iNOS and NO production in variety of cell types, including M&. In our study combination of these cytokine also was found as the most effective inducer of NO production by bone marrow derived Mg. To investigate the involvement of NO In the TNF-&iFN-y-induced expression of adhesion molecules by M# we used sodium nitroprusside as donor of NO. Our results indicate that addition of sodium nitroprusside to M#Jin dose-dependent manner inhibited both the erythrocyte binding to TNF-e/lFN-y-treated M$Jas well as ability of Mg to adhere to plastic substrata. Thus, induction of adhesion molecules by MI#Jupon TNFallFN-y stimulation appears through another mechanisms of direct NO action.
1P.3.08.25 1 Effect of Kupffer ceil phagocytosis blockade on the humorai immune response G. L&!&r’, E. Husztfk’ , G. L&r&, Jr. *, I. Kiss l, J. Olah 3, J. Srakacs I. ’ Institute of Pathophysiolog~ Albert Szent-Gyt5rgfi Medical Univemi~ Szeged, Hungaw *Deparfment of Surgery, Albert Szent-Gy&gyi Me&al Universi& Szeged, Hungary, 3L&pariment of Dermatom, Albert Szent-Gy&gyi Medical Universily, Szeged, Hungary
Introduction: TheKupffer cells, the resident macrophages of the liver comprise the largest population of macrophages in the body. The role of Kupffer cells in the aspecific resistance of the organism is well known. Because of their high functional capacity, they play important roles in the clearance of foreign substances including antigens and bacterial products. Kupffer cells have been shown to express class II antigens of the major histocompatibiiii complex and to have the capacity for antigen presentation in vita, but their regulatory roles in the induction and expression of immune response in viw have not been well defined. In the study reported here, the effects of the gadolinium chloride (GdCl&induced Kupffer cell phagocytosis blockade (Lax&, 1973, Husztik et al., 1960) were investigated on the humoral Immune responses to sheep red blood ceils (SRBC). MaterlaIrand Methods: Male CFLP mice (GOdOll6,Hungary) weighing 39-35 g were used. To induce Kupffer cell phagocytosis blockade GdCls (Prolabo) was injected iv in a dose of 0.5 mgll66 g body weight. For the primary immune response, mice were injected iv with IO’ SRBC. For the secondary immune response, animals were inoculated with the same dosage of SRBC 14 days after the primary injection. On the 4th day following immunisation, the spleens were removed and the cells were dissociated by pressing through a stainless screen. To assay humoral immune responses, the number of haemolytic plaque forming cells (Jeme and Nordln, 1963) was determined or the amount of antibody production was quantitated by the haemofytlc antibody isotope release assay (Baecher-Steppan and Kerlwilet., 1961). To measure IgG response, heat-inactivated rabbit antimouse IgG monoclonal facilitating antibodies were used. Results: The primary immune response to SRBC was significantly augmented in animals injected iv with GdCls 2, 3 or 4 days before injection of the cellular antigen. An increased secondary immune response was also observed. When GdCls was injected 2 days before the second dose of antigen, the numbers of both IgM and IgG-producing plaque folming cells were augmented. The results of the haemolytic antibody isotope release assay directly correlated with the number of antibody-producing cells measured by the plaque assay. Conclusion: Studies with 51 -Cr-labelled foreign red blood cells suggest that the augmentation of humoral immune response in GdCls-pretreated mice is a consequence of the spillover of the antigen from the liver into the spleen and other extrahepatic reticuloendothelial organs. P.3.08.26
The participation of gmnuiocytea in osteogenesis
V.V. Bazamyi, A.V. Osipenko. Ural Scientific Research lnstirute of Tmumatology and Orthopaedics, Russia, Ekaterinbutg, Russia
Introduction: Granulocytes plays an important rote in inflammatory and repair processes. However little is known about these ceils anticipation in the bone tissue regeneration. In this paper we discuss granulocytic reaction in bone formation. Materials and Methods:The model of femur lengthening after distraction osteosynthesis on dogs was used. We examined the clonogenic activity of bone marrow cells and functional characteristics of peripheral blood neutrophils (phagocytic activity, oxygen metabolism, cationic proteins level and adhesfve capacities). Reeulb: The two-phase neutrophilic reaction in dynamics of bone regeneration was demonstrated. The first phase involved postoperative inflammation and was characterized by activation of oxygen-dependent mechanisms (NBT-test). Second phase is “specifically” designed for activation of osteoblastic processes. The increase of the cationic protein level in this period was noticed. Clonogenic activity of granulocytic progenitor cells in normal osteogenesis did not changes. Adhesive capashy and the efffciency of doning of granulocyfic progenitor cells in impaired bone formation differed from normal osteogenesis. Conclusion: We demonstrated certain changes of neutrophil status in normal and disturbed bone fonatfon. It proves participation of granulocytes in osteogenesis regulation.
23 June 1997 - Poster presentations
Phagocyte activation adfunction
P.3.08.23
Fosslbie sequence of events resuiting in resolution of infection with Francisella fdamnsis in mice
A. Macela ‘, M. Kroca I.*, L. Hemychova I, H. Kovamva ‘, J. Stulik ‘, 2. Krocova I. ’ Institute for Immunotog~ Pmkyne Military Medical Academ)! 500 01 Hmdec Kmlove, Czech Republic, 2Dept. Micmbiol., National LIefence Research Establishment, S-901 82 Umea, Sweden Introduction: Regulatory events dudng the immune response against intracellular bacterial pathogens are commonly understood. On the other hand, the inductive phase and decisive mechanisms operating in the course of primary response remain unclear. Our effort was focused on review of possible inductive interactions leading to the resolution of primary infection of mice with intracellular bacterial pathogen Ffancise//a tulafensls and resulting in ultimate protective immunity. Matertals and Methods: Subcutaneous infection of mice with Fran&e//a tularensis strain LVS in sublethal doses was used in this study. Several in vitro reactions of antibody with antigen, test of cytotoxicity of several populations of effector cells based on the release of 51chromium from target cells, and the levels of extracellular cytokines measured by commercial ELISA sets were utilized for screening of immunological status of host mice. ResuRs: Primary immune response against intracellular bacterial pathogen Fmncise//a Warensis can be induced by possible direct primary interactions of microbes with macrophages, NK cells, and B cells. These primary interactions lead to the production of several cytokines. Among them, the early production of IL-10 is probably responsible for increasing lytic activity of NK cells. Concomitant production of IFN-gamma is responsible for priming of macrophages for metabolic and functional activation. Simultaneously, with the production of TNF-alpha and IL-6 by macrophages, and the presence of specific antitularemic antibodies, the increase of number of bacteria in organs of infected mice was diminished. According to the cytokine profile (11-2,IL-4) Thl and Th2 branches of immunoregulatory mechanism are operating in this stage of infection. In the course of the highest activity of regulatory lymphocytes, cytotoxic macrophages and the effector cells using specific antibodies for extracellular FmnciseMatularensis killing occur. Together with the appearance of above-mentioned activities, the absolute decrease of microbes in organs of infected mice was evident. Conclusion: In summary, our results demonstrate that for resolution of primary Fmncisella tolamnsis infection of mice, the early production of IL-IO and TNF-alpha play most important role. The presence of antitularemic antibodies has dominant role in effective elimination of microbes from tissues of infected mice.
P.3.08.24
TNF-ar plus IFNq induce expression of siaioadhesin receptor by stromai bone marrow macrophages (M#)
S.A. Kusmartsev’, J. Navarro’, I. Angulo2, M. Danylets’, J.L. Subiza*. ‘Lab Siomodets of Tomsk Research Center Tomsk, Russia, * Servicio de lnmunologia Hospital Univemitario San Caries, Madrid, Spain Stromal murine Mg in adult bone marrow and lymphoid organs express a specific adhesion receptor which interact with sialited structures on developing myeloid cells and also on sheep erythrocytes. To test whether combination of TNF-n plus IFN-y, that is known as inductor differentiation of myeloid cells, could synergize for induction of sialoadhesin receptor, we used resetting assay with 51Cr-labeled sheep erythrocytes to detect functional surface expression of sialoadhesin on bone marrow derived M+. Thls method was combined with crystal violet assay and reverse centrlfugatfon. Our result showed that treatment of bone marrow M# by TNFa or IFN-y alone for 24 h, did not induce of erythrocyte binding to MI$ as compared with control. The highest binding of 51Cr-labeled erythmcytes observed after culturing of M+ in presence TNFa plus IFN-y (4 to 5-fold increase in 3 separate experfments). Addition of IL-1 to this combination did not enhance observed effect. Using IL-1 alone, as well as LPS or M-CSF had a weaker stimulation effect. As showed experiments with blocking anti-sialoadhesin monoclonal antibodies and also with neuramlnidasae-treated erythrocytes this TNF&lFN-y- inducing effect was mediated by siafoadhesln. We also found that treatment of M# with this cytokine combination for 24 h stimulated attaching of these cells to plastic substrata. Taken together our data demonstrate that TNF-a plus IFN-y has strong inducing effect on adhesion molecule expression by bone marrow M&. On the other hand, combination of TNFa and IFN-y is well known as inductor expression of iNOS and NO production in variety of cell types, including M&. In our study combination of these cytokine also was found as the most effective inducer of NO production by bone marrow derived Mg. To investigate the involvement of NO In the TNF-&iFN-y-induced expression of adhesion molecules by M# we used sodium nitroprusside as donor of NO. Our results indicate that addition of sodium nitroprusside to M#Jin dose-dependent manner inhibited both the erythrocyte binding to TNF-e/lFN-y-treated M$Jas well as ability of Mg to adhere to plastic substrata. Thus, induction of adhesion molecules by MI#Jupon TNFallFN-y stimulation appears through another mechanisms of direct NO action.
1P.3.08.25 1 Effect of Kupffer ceil phagocytosis blockade on the humorai immune response G. L&!&r’, E. Husztfk’ , G. L&r&, Jr. *, I. Kiss l, J. Olah 3, J. Srakacs I. ’ Institute of Pathophysiolog~ Albert Szent-Gyt5rgfi Medical Univemi~ Szeged, Hungaw *Deparfment of Surgery, Albert Szent-Gy&gyi Me&al Universi& Szeged, Hungary, 3L&pariment of Dermatom, Albert Szent-Gy&gyi Medical Universily, Szeged, Hungary
Introduction: TheKupffer cells, the resident macrophages of the liver comprise the largest population of macrophages in the body. The role of Kupffer cells in the aspecific resistance of the organism is well known. Because of their high functional capacity, they play important roles in the clearance of foreign substances including antigens and bacterial products. Kupffer cells have been shown to express class II antigens of the major histocompatibiiii complex and to have the capacity for antigen presentation in vita, but their regulatory roles in the induction and expression of immune response in viw have not been well defined. In the study reported here, the effects of the gadolinium chloride (GdCl&induced Kupffer cell phagocytosis blockade (Lax&, 1973, Husztik et al., 1960) were investigated on the humoral Immune responses to sheep red blood ceils (SRBC). MaterlaIrand Methods: Male CFLP mice (GOdOll6,Hungary) weighing 39-35 g were used. To induce Kupffer cell phagocytosis blockade GdCls (Prolabo) was injected iv in a dose of 0.5 mgll66 g body weight. For the primary immune response, mice were injected iv with IO’ SRBC. For the secondary immune response, animals were inoculated with the same dosage of SRBC 14 days after the primary injection. On the 4th day following immunisation, the spleens were removed and the cells were dissociated by pressing through a stainless screen. To assay humoral immune responses, the number of haemolytic plaque forming cells (Jeme and Nordln, 1963) was determined or the amount of antibody production was quantitated by the haemofytlc antibody isotope release assay (Baecher-Steppan and Kerlwilet., 1961). To measure IgG response, heat-inactivated rabbit antimouse IgG monoclonal facilitating antibodies were used. Results: The primary immune response to SRBC was significantly augmented in animals injected iv with GdCls 2, 3 or 4 days before injection of the cellular antigen. An increased secondary immune response was also observed. When GdCls was injected 2 days before the second dose of antigen, the numbers of both IgM and IgG-producing plaque folming cells were augmented. The results of the haemolytic antibody isotope release assay directly correlated with the number of antibody-producing cells measured by the plaque assay. Conclusion: Studies with 51 -Cr-labelled foreign red blood cells suggest that the augmentation of humoral immune response in GdCls-pretreated mice is a consequence of the spillover of the antigen from the liver into the spleen and other extrahepatic reticuloendothelial organs. P.3.08.26
The participation of gmnuiocytea in osteogenesis
V.V. Bazamyi, A.V. Osipenko. Ural Scientific Research lnstirute of Tmumatology and Orthopaedics, Russia, Ekaterinbutg, Russia
Introduction: Granulocytes plays an important rote in inflammatory and repair processes. However little is known about these ceils anticipation in the bone tissue regeneration. In this paper we discuss granulocytic reaction in bone formation. Materials and Methods:The model of femur lengthening after distraction osteosynthesis on dogs was used. We examined the clonogenic activity of bone marrow cells and functional characteristics of peripheral blood neutrophils (phagocytic activity, oxygen metabolism, cationic proteins level and adhesfve capacities). Reeulb: The two-phase neutrophilic reaction in dynamics of bone regeneration was demonstrated. The first phase involved postoperative inflammation and was characterized by activation of oxygen-dependent mechanisms (NBT-test). Second phase is “specifically” designed for activation of osteoblastic processes. The increase of the cationic protein level in this period was noticed. Clonogenic activity of granulocytic progenitor cells in normal osteogenesis did not changes. Adhesive capashy and the efffciency of doning of granulocyfic progenitor cells in impaired bone formation differed from normal osteogenesis. Conclusion: We demonstrated certain changes of neutrophil status in normal and disturbed bone fonatfon. It proves participation of granulocytes in osteogenesis regulation.