The Experts below are selected from a list of 174 Experts worldwide ranked by ideXlab platform
Hidetoshi Inoko - One of the best experts on this subject based on the ideXlab platform.
-
Decrease in CD93 (C1qRp) Expression in a Human Monocyte‐Like Cell Line (U937) Treated with Various Apoptosis‐Inducing Chemical Substances
Microbiology and Immunology, 2020Co-Authors: Nobunao Ikewaki, H Tamauchi, Hidetoshi InokoAbstract:Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by Cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various Cells, particularly in granulocytes and Monocytes. We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured Cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid Cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human Monocyte-Like Cell line (U937) treated with various apoptosis-inducing chemical substances: an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (–) Cells and Annexin V positive (+)/7AAD positive (+) Cells, respectively, in U937 Cells after 6 hr of treatment. The modulation of CD93 expression on U937 Cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (–) Cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated Cell sorter (FACS). The mean fluorescence intensity (MFI) of the Cells that stained positive for CD93 mAb (mNI-11) among the treated U937 Cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 β; LFA-1β) and CD54 (interCellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 Cells identified as Annexin V positive (+)/7AAD negative (—) Cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 Cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 Cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 Cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 Cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 Cells. Importantly, however, the U937 Cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 Cells with apoptotic properties, and that the decrease in CD93 expression on U937 Cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid Cells.
-
decrease in cd93 c1qrp expression in a human Monocyte Like Cell line u937 treated with various apoptosis inducing chemical substances
Microbiology and Immunology, 2007Co-Authors: Nobunao Ikewaki, H Tamauchi, Hidetoshi InokoAbstract:Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by Cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various Cells, particularly in granulocytes and Monocytes. We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured Cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid Cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human Monocyte-Like Cell line (U937) treated with various apoptosis-inducing chemical substances: an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (–) Cells and Annexin V positive (+)/7AAD positive (+) Cells, respectively, in U937 Cells after 6 hr of treatment. The modulation of CD93 expression on U937 Cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (–) Cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated Cell sorter (FACS). The mean fluorescence intensity (MFI) of the Cells that stained positive for CD93 mAb (mNI-11) among the treated U937 Cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 β; LFA-1β) and CD54 (interCellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 Cells identified as Annexin V positive (+)/7AAD negative (—) Cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 Cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 Cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 Cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 Cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 Cells. Importantly, however, the U937 Cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 Cells with apoptotic properties, and that the decrease in CD93 expression on U937 Cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid Cells.
-
Regulation of CD93 Cell surface expression by protein kinase C isoenzymes
Microbiol Immunol, 2006Co-Authors: N Ikewaki, Jerzy K. Kulski, Hidetoshi InokoAbstract:Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by Cells with a myeloid lineage, endothelial Cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intraCellular molecular events responsible for the regulation of its expression on the Cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the Cell surface of a human Monocyte-Like Cell line (U937), a human NK-Like Cell line (KHYG-1), and a human umbilical vein endothelial Cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed Cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 Cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 Cells and by both Rottlerin and Go6976 in the HUV-EC-C Cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the Cell surface of all three Cell-lines and induced interleukin-8 (IL-8) production by the U937 Cells and interferon-gamma (IFN-gamma) production by the KHYG-1 Cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective Cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 Cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these Cells involves the PKC isoenzymes.
-
A Microfilament Formation Inhibitor, Cytochalasin Strongly Enhances the Low-Affinity Fc ε Receptor II (CD23) Expression on the Human Monocyte-Like Cell Line, U937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc ε receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
-
a microfilament formation inhibitor cytochalasin strongly enhances the low affinity fc e receptor ii cd23 expression on the human Monocyte Like Cell line u937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc e receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
Nobunao Ikewaki - One of the best experts on this subject based on the ideXlab platform.
-
Decrease in CD93 (C1qRp) Expression in a Human Monocyte‐Like Cell Line (U937) Treated with Various Apoptosis‐Inducing Chemical Substances
Microbiology and Immunology, 2020Co-Authors: Nobunao Ikewaki, H Tamauchi, Hidetoshi InokoAbstract:Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by Cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various Cells, particularly in granulocytes and Monocytes. We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured Cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid Cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human Monocyte-Like Cell line (U937) treated with various apoptosis-inducing chemical substances: an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (–) Cells and Annexin V positive (+)/7AAD positive (+) Cells, respectively, in U937 Cells after 6 hr of treatment. The modulation of CD93 expression on U937 Cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (–) Cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated Cell sorter (FACS). The mean fluorescence intensity (MFI) of the Cells that stained positive for CD93 mAb (mNI-11) among the treated U937 Cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 β; LFA-1β) and CD54 (interCellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 Cells identified as Annexin V positive (+)/7AAD negative (—) Cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 Cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 Cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 Cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 Cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 Cells. Importantly, however, the U937 Cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 Cells with apoptotic properties, and that the decrease in CD93 expression on U937 Cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid Cells.
-
decrease in cd93 c1qrp expression in a human Monocyte Like Cell line u937 treated with various apoptosis inducing chemical substances
Microbiology and Immunology, 2007Co-Authors: Nobunao Ikewaki, H Tamauchi, Hidetoshi InokoAbstract:Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by Cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various Cells, particularly in granulocytes and Monocytes. We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured Cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid Cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human Monocyte-Like Cell line (U937) treated with various apoptosis-inducing chemical substances: an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (–) Cells and Annexin V positive (+)/7AAD positive (+) Cells, respectively, in U937 Cells after 6 hr of treatment. The modulation of CD93 expression on U937 Cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (–) Cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated Cell sorter (FACS). The mean fluorescence intensity (MFI) of the Cells that stained positive for CD93 mAb (mNI-11) among the treated U937 Cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 β; LFA-1β) and CD54 (interCellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 Cells identified as Annexin V positive (+)/7AAD negative (—) Cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 Cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 Cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 Cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 Cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 Cells. Importantly, however, the U937 Cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 Cells with apoptotic properties, and that the decrease in CD93 expression on U937 Cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid Cells.
-
A Microfilament Formation Inhibitor, Cytochalasin Strongly Enhances the Low-Affinity Fc ε Receptor II (CD23) Expression on the Human Monocyte-Like Cell Line, U937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc ε receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
-
a microfilament formation inhibitor cytochalasin strongly enhances the low affinity fc e receptor ii cd23 expression on the human Monocyte Like Cell line u937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc e receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
-
a novel monoclonal antibody mni 58a against the α chain of leukocyte function associated antigen 1 lfa 1 blocks the homotypic Cell aggregation and actively regulates morphological changes in the ohorbol myristate acetate pma activated human Monocyte
Tissue Antigens, 1996Co-Authors: Nobunao Ikewaki, Akira Yamada, A Sonoda, Hidetoshi InokoAbstract:: A monoclonal antibody (mAb), designated mNI-58A, was produced by immunizing mice with the lipopolysaccharide (LPS)-stimulated Monocyte-Like Cell line, U937. The antigen defined by mNI-58A was widely expressed on various lymphoid Cells and all Cell lines examined except the erythroid Cell line, K562. When the reactive patterns between mNI-58A and the mAbs to various human differentiation antigens (CD11a, CD11b, CD11c, CD14, CD16, CD18, CD23, CD28, CD29, CD31, CD43, CD44, CD45RA, CD50, CD54, CD58, CD80, CD102, CD106, HLA-class I and-class II antigen) were compared, that of mNI-58A was found to be similar to those of the leukocyte function-associated antigen-1 (LFA-1) mAbs. Using a competitive immunofluorescence binding assay it was found that the preincubation with one of the CD11a mAbs, 2F12 completely blocked the subsequent binding of mNI-58A. mNI-58A prevented the homotypic Cell aggregation of the phorbol myristate acetate (PMA)-activated U937 Cells (referred to as PMA-U937) and PMA-activated Epstein-Barr virus (EBV)-transformed B Cell lines, B-85 and Mann. mNI-58A markedly induced the spread formation of the PMA-U937 Cells following this blocking of the homotypic Cell aggregation, whereas 2F12 did not under the same condition. The spread formation induced by mNI-58A was completely blocked by cytochalasin B (CyB), cytochalasin D (CyD), cycloheximide (CHX) or protein kinase C inhibitors, sphingosine and H-7. The U937 Cells markedly adhered to the tumor necrosis factor-alpha (TNF-alpha)-stimulated human umbilical vein endothelial Cells (HUVECs) and also to the extraCellular matrix protein, fibronectin, but mNI-58A did not enhance or block these adhesion process. mNI-58A precipitated two glycoproteins with molecular weight 180 kDa and 95 kDa as determined by SDS-PAGE analysis, which were identical to the LFA-alpha (CD11a) and beta (CD18) chains of leukocyte integrin precipitated by the CD11a mAbs, respectively. Sequential immunoprecipitation studies using the CD11a mAb (2F12) also indicate that mNI-58A recognizes an epitope on the alpha-chain of the LFA-1 molecule. The ability of mNI-58A to block the PMA-U937 Cells and to induce the spread formation of these Cells suggests that mNI-58A is a novel mAb reacting with an epitope on the alpha-chain of LFA-1 different from those recognized with the existing CD11a mAbs.
Timothy D Bigby - One of the best experts on this subject based on the ideXlab platform.
-
lipopolysaccharide down regulates the leukotriene c4 synthase gene in the Monocyte Like Cell line thp 1
Journal of Immunology, 2003Co-Authors: Kenneth J Serio, Scott C Johns, Craig R Hodulik, Timothy D BigbyAbstract:We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC 4 synthase expression in mononuclear phagocytes. Conditioning of the Monocyte-Like Cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC 4 release. The putative LPS receptor, Toll-Like receptor 4, was expressed in THP-1 Cells. LPS down-regulated LTC 4 synthase mRNA in THP-1 Cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated Cells with LPS resulted in no change in the rate of LTC 4 synthase mRNA decay. LPS treatment of THP-1 Cells, transiently transfected with a LTC 4 synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-α and inhibition of mitogen-activated protein kinase kinase/extraCellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of Cells with a Toll-Like receptor 4-blocking Ab and an inhibitor of NF-κB activation resulted in inhibition of the LPS effect, while activation of NF-κB and p50/p65 overexpression down-regulated the LTC 4 synthase gene. LPS down-regulates cysteinyl LT release and LTC 4 synthase gene expression in mononuclear phagocytes by an NF-κB-mediated mechanism.
-
sp1 and sp3 function as key regulators of leukotriene c 4 synthase gene expression in the Monocyte Like Cell line thp 1
American Journal of Respiratory Cell and Molecular Biology, 2000Co-Authors: Kenneth J Serio, Craig R Hodulik, Timothy D BigbyAbstract:The goal of this study was to examine the mechanisms of leukotriene C4 (LTC4) synthase gene expression in mononuclear phagocytes. Transfection of the Monocyte-Like Cell line THP-1 with LTC4 synthase promoter–reporter constructs demonstrated that the first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity. Deletion analysis revealed that the region between − 92 and − 23 bp, which contains a signal protein (Sp)1 consensus site at − 42 to − 37 bp, mediated an 11.5-fold increase in reporter activity. Using a probe from − 56 to − 17 bp, electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and THP-1 and HeLa nuclear extracts bind to this region. Binding was eliminated by mutation of the Sp1 consensus site. Supershift EMSAs using anti-Sp1 and anti-Sp3 antibodies demonstrated that these Sp family members bind to the region. Transfection of the Sp-deficient Drosophila SL-2 Cell line with a construct containing the − 92 to − 23 bp promoter region and Sp expression vectors reve...
-
tgf beta increases leukotriene c4 synthase expression in the Monocyte Like Cell line thp 1
Journal of Immunology, 1999Co-Authors: C. A. Riddick, Kenneth J Serio, Craig R Hodulik, W. L. Ring, Mark S Regan, Timothy D BigbyAbstract:The goal of this study was to determine whether cytokines modulate leukotriene C4 (LTC4) synthase expression in mononuclear phagocytes. A panel of cytokines was surveyed for changes in LTC4 synthase mRNA in THP-1 Cells. TGF-β1, -2, and -3 had significant stimulatory effects. The addition of TGF-β resulted in a time-dependent increase in LTC4 synthase mRNA at 6 h, which persisted through 48 h. Furthermore, this conditioning resulted in an increase in immunoreactive protein for LTC4 synthase through 7 days. TGF-β conditioning of Cells resulted in a time- and dose-dependent increase in stimulated LTC4 synthase activity. Following transient transfection of THP-1 Cells with a promoter-reporter construct containing 1.2 kb of the LTC4 synthase promoter, TGF-β treatment resulted in a 2-fold increase in reporter activity. Conditioning with TGF-β did not prolong the half-life of LTC4 synthase mRNA, as assessed by RNase protection assays in actinomycin D-treated Cells. Cycloheximide exposure experiments revealed that new protein synthesis was not required for the observed stimulatory effect of TGF-β on LTC4 synthase mRNA. We conclude that LTC4 synthase expression is increased at a transcriptional level by TGF-β in mononuclear phagocytes.
-
Activated lymphocytes increase expression of 5-lipoxygenase and its activating protein in THP-1 Cells.
American Journal of Physiology-cell Physiology, 1997Co-Authors: W. L. Ring, C. A. Riddick, J. R. Baker, Christopher K. Glass, Timothy D BigbyAbstract:The aim of this study was to investigate the regulation of the 5-lipoxygenase pathway of arachidonic acid metabolism by lymphocytes using the Monocyte-Like Cell line, THP-1. When THP-1 Cells were i...
H Tamauchi - One of the best experts on this subject based on the ideXlab platform.
-
Decrease in CD93 (C1qRp) Expression in a Human Monocyte‐Like Cell Line (U937) Treated with Various Apoptosis‐Inducing Chemical Substances
Microbiology and Immunology, 2020Co-Authors: Nobunao Ikewaki, H Tamauchi, Hidetoshi InokoAbstract:Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by Cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various Cells, particularly in granulocytes and Monocytes. We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured Cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid Cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human Monocyte-Like Cell line (U937) treated with various apoptosis-inducing chemical substances: an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (–) Cells and Annexin V positive (+)/7AAD positive (+) Cells, respectively, in U937 Cells after 6 hr of treatment. The modulation of CD93 expression on U937 Cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (–) Cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated Cell sorter (FACS). The mean fluorescence intensity (MFI) of the Cells that stained positive for CD93 mAb (mNI-11) among the treated U937 Cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 β; LFA-1β) and CD54 (interCellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 Cells identified as Annexin V positive (+)/7AAD negative (—) Cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 Cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 Cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 Cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 Cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 Cells. Importantly, however, the U937 Cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 Cells with apoptotic properties, and that the decrease in CD93 expression on U937 Cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid Cells.
-
decrease in cd93 c1qrp expression in a human Monocyte Like Cell line u937 treated with various apoptosis inducing chemical substances
Microbiology and Immunology, 2007Co-Authors: Nobunao Ikewaki, H Tamauchi, Hidetoshi InokoAbstract:Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by Cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various Cells, particularly in granulocytes and Monocytes. We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured Cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid Cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human Monocyte-Like Cell line (U937) treated with various apoptosis-inducing chemical substances: an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (–) Cells and Annexin V positive (+)/7AAD positive (+) Cells, respectively, in U937 Cells after 6 hr of treatment. The modulation of CD93 expression on U937 Cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (–) Cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated Cell sorter (FACS). The mean fluorescence intensity (MFI) of the Cells that stained positive for CD93 mAb (mNI-11) among the treated U937 Cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 β; LFA-1β) and CD54 (interCellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 Cells identified as Annexin V positive (+)/7AAD negative (—) Cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 Cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 Cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 Cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 Cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 Cells. Importantly, however, the U937 Cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 Cells with apoptotic properties, and that the decrease in CD93 expression on U937 Cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid Cells.
-
A Microfilament Formation Inhibitor, Cytochalasin Strongly Enhances the Low-Affinity Fc ε Receptor II (CD23) Expression on the Human Monocyte-Like Cell Line, U937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc ε receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
-
a microfilament formation inhibitor cytochalasin strongly enhances the low affinity fc e receptor ii cd23 expression on the human Monocyte Like Cell line u937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc e receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
-
modulation of Cell surface antigens and regulation of phagocytic activity mediated by cd11b in the Monocyte Like Cell line u937 in response to lipopolysaccharide
Tissue Antigens, 1993Co-Authors: Nabunae Ikewaki, Hidetoshi Inoko, H TamauchiAbstract:: Modulation of the Cellular antigens and regulation of the phagocytic activity of the Monocyte-Like Cell line U937 after culture with lipopolysaccharide (LPS) were investigated. CD14 expression was induced on the surface of the U937 Cells after 48 h of culture with LPS and then they became adhesive with numerous filamentous filopodia extruded on the Cell surface, exhibiting the enhanced expression of CD16 and CD23, the activation Cell surface markers for differentiation into macrophage. However, no induction or enhancement of the Cell surface expression was observed with respect to CD11b, CD18, HLA-A, B, C, HLA-DR, DQ, DP or CD57. These U937 Cells also acquired the ability to produce superoxide anions and to phagocytose the Salmonella enteritidis strain, 116–54. This phagocytosis was inhibited by the anti-CD11b monoclonal antibodies, but not by the anti-CD14, anti-CD16, anti-CD18, anti-CD23, anti-HLA-A, B, C or anti-HLA-DR monoclonal antibodies. These findings indicate that the phagocytic activity against Salmonella enteritidis 116–54 induced by LPS is mediated mainly via the CD11b molecule, but is not associated with the increased expression of CD11b. Puromycin and cycloheximide, inhibitors of protein synthesis, or a divalent cation-chelating agent, EDTA completely inhibited this phagocytic activity. Interestingly, EDTA was found to suppress specifically the CD11b expression on the U937 Cells cultured with LPS. No phagocytic activity was induced when the U937 Cells cultured with LPS were incubated at 4°C, but restored to the control level when shifted up to 37°C. This experimental system provides an exCellent model for the investigation of the molecular mechanism underlying modulation of the Cell surface antigens and regulation of the phagocytic activity mediated by CD11b in the U937 Cells in response to LPS.
Akira Yamada - One of the best experts on this subject based on the ideXlab platform.
-
A Microfilament Formation Inhibitor, Cytochalasin Strongly Enhances the Low-Affinity Fc ε Receptor II (CD23) Expression on the Human Monocyte-Like Cell Line, U937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc ε receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
-
a microfilament formation inhibitor cytochalasin strongly enhances the low affinity fc e receptor ii cd23 expression on the human Monocyte Like Cell line u937
Journal of Clinical Immunology, 2000Co-Authors: Nobunao Ikewaki, H Tamauchi, Akira Yamada, Masahito Aoki, Ryuichi Yamamoto, Atsushi Sawada, Hidetoshi InokoAbstract:Enhancement of the low-affinity Fc e receptor (CD23) expression by cytochalasin was analyzed on the human MonocyteLike Cell line, U937. The CD23 expression on the U937 Cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 Cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 Cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but Cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 Cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 Cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 Cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 Cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.
-
a novel monoclonal antibody mni 58a against the α chain of leukocyte function associated antigen 1 lfa 1 blocks the homotypic Cell aggregation and actively regulates morphological changes in the ohorbol myristate acetate pma activated human Monocyte
Tissue Antigens, 1996Co-Authors: Nobunao Ikewaki, Akira Yamada, A Sonoda, Hidetoshi InokoAbstract:: A monoclonal antibody (mAb), designated mNI-58A, was produced by immunizing mice with the lipopolysaccharide (LPS)-stimulated Monocyte-Like Cell line, U937. The antigen defined by mNI-58A was widely expressed on various lymphoid Cells and all Cell lines examined except the erythroid Cell line, K562. When the reactive patterns between mNI-58A and the mAbs to various human differentiation antigens (CD11a, CD11b, CD11c, CD14, CD16, CD18, CD23, CD28, CD29, CD31, CD43, CD44, CD45RA, CD50, CD54, CD58, CD80, CD102, CD106, HLA-class I and-class II antigen) were compared, that of mNI-58A was found to be similar to those of the leukocyte function-associated antigen-1 (LFA-1) mAbs. Using a competitive immunofluorescence binding assay it was found that the preincubation with one of the CD11a mAbs, 2F12 completely blocked the subsequent binding of mNI-58A. mNI-58A prevented the homotypic Cell aggregation of the phorbol myristate acetate (PMA)-activated U937 Cells (referred to as PMA-U937) and PMA-activated Epstein-Barr virus (EBV)-transformed B Cell lines, B-85 and Mann. mNI-58A markedly induced the spread formation of the PMA-U937 Cells following this blocking of the homotypic Cell aggregation, whereas 2F12 did not under the same condition. The spread formation induced by mNI-58A was completely blocked by cytochalasin B (CyB), cytochalasin D (CyD), cycloheximide (CHX) or protein kinase C inhibitors, sphingosine and H-7. The U937 Cells markedly adhered to the tumor necrosis factor-alpha (TNF-alpha)-stimulated human umbilical vein endothelial Cells (HUVECs) and also to the extraCellular matrix protein, fibronectin, but mNI-58A did not enhance or block these adhesion process. mNI-58A precipitated two glycoproteins with molecular weight 180 kDa and 95 kDa as determined by SDS-PAGE analysis, which were identical to the LFA-alpha (CD11a) and beta (CD18) chains of leukocyte integrin precipitated by the CD11a mAbs, respectively. Sequential immunoprecipitation studies using the CD11a mAb (2F12) also indicate that mNI-58A recognizes an epitope on the alpha-chain of the LFA-1 molecule. The ability of mNI-58A to block the PMA-U937 Cells and to induce the spread formation of these Cells suggests that mNI-58A is a novel mAb reacting with an epitope on the alpha-chain of LFA-1 different from those recognized with the existing CD11a mAbs.
-
A novel monoclonal antibody mNI‐58A against the α‐chain of leukocyte function‐associated antigen‐1 (LFA‐1) blocks the homotypic Cell aggregation and actively regulates morphological changes in the ohorbol myristate acetate (PMA)‐activated human monoc
Tissue Antigens, 1996Co-Authors: Nobunao Ikewaki, Akira Yamada, A Sonoda, Hidetoshi InokoAbstract:A monoclonal antibody (mAb), designated mNI-58A, was produced by immunizing mice with the lipopolysaccharide (LPS)-stimulated Monocyte-Like Cell line, U937. The antigen defined by mNI-58A was widely expressed on various lymphoid Cells and all Cell lines examined except the erythroid Cell line, K562. When the reactive patterns between mNI-58A and the mAbs to various human differentiation antigens (CD11a, CD11b, CD11c, CD14, CD16, CD18, CD23, CD28, CD29, CD31, CD43, CD44, CD45RA, CD50, CD54, CD58, CD80, CD102, CD106, HLA-class I and-class II antigen) were compared, that of mNI-58A was found to be similar to those of the leukocyte function-associated antigen-1 (LFA-1) mAbs. Using a competitive immunofluorescence binding assay it was found that the preincubation with one of the CD11a mAbs, 2F12 completely blocked the subsequent binding of mNI-58A. mNI-58A prevented the homotypic Cell aggregation of the phorbol myristate acetate (PMA)-activated U937 Cells (referred to as PMA-U937) and PMA-activated Epstein-Barr virus (EBV)-transformed B Cell lines, B-85 and Mann. mNI-58A markedly induced the spread formation of the PMA-U937 Cells following this blocking of the homotypic Cell aggregation, whereas 2F12 did not under the same condition. The spread formation induced by mNI-58A was completely blocked by cytochalasin B (CyB), cytochalasin D (CyD), cycloheximide (CHX) or protein kinase C inhibitors, sphingosine and H-7. The U937 Cells markedly adhered to the tumor necrosis factor-alpha (TNF-alpha)-stimulated human umbilical vein endothelial Cells (HUVECs) and also to the extraCellular matrix protein, fibronectin, but mNI-58A did not enhance or block these adhesion process. mNI-58A precipitated two glycoproteins with molecular weight 180 kDa and 95 kDa as determined by SDS-PAGE analysis, which were identical to the LFA-alpha (CD11a) and beta (CD18) chains of leukocyte integrin precipitated by the CD11a mAbs, respectively. Sequential immunoprecipitation studies using the CD11a mAb (2F12) also indicate that mNI-58A recognizes an epitope on the alpha-chain of the LFA-1 molecule. The ability of mNI-58A to block the PMA-U937 Cells and to induce the spread formation of these Cells suggests that mNI-58A is a novel mAb reacting with an epitope on the alpha-chain of LFA-1 different from those recognized with the existing CD11a mAbs.