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IL-1α regulates interleukin 2 gene expression in murine thymoma cells by a post-initiation mechanism
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MODULATION OF TUMOR ASSOCIATED MACROPHAGE ACTIVITY BY RECOMBINANT INTERFERON-GAMMA. M.J. Thomassen, T.W. Rice, H.P. Wiedemann, D.P. Meeker and M. Ahmad. Cleveland Clinic Foundation, Cleveland, OH 44195. We evaluated the cytotoxic response of tumor associated macrophages (TAMS) t" recombinant interferon-gamma (Biogen). TAMS were recovered from resected tumor tissue of 5 patients with primary lung cancer. From 4 of these patients, macrophages were also isolated from """tumor tissue. After digestion of tissue with a mixture of collagenase and DNase, the cell suspension was adhered to plastic. The resulting cell population was >90% macrophages as evidenced by morphology and nonspecific esterase staining. Macrophage yields from 7-16g of tumor tissue varied between 0.8 - 6.1 x IO6 macronhaees. From 6-16~ of """tumor tissue. 0.7 - 10.8 x lo6 macro;ha;es were obtain:d. Tumoricidal activity was assessed using 'H-thymidine labelled human tumor cells (SK-MEL-28, CRL 1718). TAMS from only l/5 tumors demonstrated spontaneous tumoricidal activity, but after exposure to interferon-gamma the TAMS from the remaining 4 tumors also demonstrated cytotoxicity against tumor targets (44.0 5 5.8, mean % cytotoxicity 2 SEM). Macrophages from """tumor tissue demonstrated similar cytotoxicity (39.5 ? 6.7) after exposure to interferon-gamma. These results suggest that the tumoricidal activity of both TAMs and """tumor tissue macrophages may be enhanced by interferon-gamma.
DIFFERENTIAL EFFECTS OF TRANSFORMING GROWTH FACTOR-B (TGF-!3) AND EPIDERMAL GROWTH FACTOR (EGF) ON THE CELL CYCLE OF CULTURED RABBIT ARTICULAR CHONDROCYTES. D.Vivieq*, E.Lebrun**, P.GalCra*+, G.Lovak* and J-P.Puiol*. Laboratoire de Biochimie du Tissu Conjonctif*, Centre de Transfusion Sanguine**, CHU C&e de Nacre, 14033 Caen Cedex, France. In presence of 2 or 10% FCS. TGF-B at 0.1. 1 and 2 “g/ml had no effect on cell proliferation after 24-h exposure. By flow cytometric analysis we demonstrated that a high proportion of cells were arrested in late S-phase at this time. In both serum concentrations, a disappearance of the blocked cells occurred at 48 h after TGF-I3 addition. At this time, an increase of cell proliferation was only seen for 10% FCS. This suggests that TGF-!3 exerta a stimulatory effect on the replication in optimal concentration of serum. We compared the effect of TGF-!3 to that of EGF. In both quiescent (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. without arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases. [3H]thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-!3 than for EGF. Although both factors induce similar increases in cell number at 48 h in 10 % FCS-containing medium, these proliferative effects were due to different actions on the cell cycle.
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ROLE OF LYMPHOCYTE FUNCTION-ASSOCIATED ANTIGEN-l (LFA-1) IN THE MIGRATION OF T CELLS TO LYMPHOCYTE CHEMOTACTIC FACTOR (LCF) AND IL-Z. M. Vachula, C.W. Smith. J. Potter. D.C. Anderson. D.E. Van Eous. Applied Sciences, Baxter Healthcare Cot-p., Round Lake, IL 60073; Baylor Cal. of Med., Houston, TX 77046; Univ. of New Mexico, Albuquerque, NM 87131. The role of LFA-1 in the chemotactic response of human T cells was examined. LCF, purified from concanavalin A stimulated peripheral blood mononuclear cells, and rIL-2 were used to stimulate T cell migration in blind well chemotaxis chambers. Chemotaxis was measured by the leading front assay using T cells prepared by E-rosetting (>90% CD3+ as determined by flow cytometry). The role of LFA-1 in migration was evaluated using antibodies to the LFA-1 a chain, CDlla (TS1/22) and I3 chain, CD18 (TS1/18). T cell chemotaxis in response to LCF and IL-2 was significantly suppressed by TS1/18 antibody and to a lesser degree by TS1/22. Neither antibody suppressed random T cell migration. Control anti-CD4 monoclonal antibody of the same IgGl isotype as the anti-LFA antibodies did not inhibit chemotaxis or random migration. Additional studies to evaluate the adherence and migration of T cells through IL-1 stimulated human umbilical vein endothelial cell monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration but failed to inhibit adherence. These studies show that LFA-1 plays a role in the migration of T cells through endothelial cells and that the R chain of LFA-1 is important in the in vitro migration of T cells in response to LCF and IL-2.
PHORBOL ESTERS CAN PERSISTENTLY REPLACE INTERLEUKIN-3 (IL-3) FOR THE GROWTH OF A IL-3-DEPENDENT CELL LINE. K. Ways, R. Riddle, L. Steelman, and J. McCubrey. East Carolina Univ. Sch. of Medicine, Greenville, NC. 27858. FDC-Pl is a" IL-3-dependent cell line which normally dies in the absence of IL-3. We have isolated a variant cell line that grows continuously in the presence of phorbol myristate acetate (PMA). This variant cell line (FD-PMA) has been maintained in culture for 10 months and optimal cell growth is achieved in PMA rather than in IL-3. LFA-1, Mac-l, and Mac-2 were readily detected on the cell surface of FD-PMA, however these antigens were not detected No lymphokine gene expression on the parental cell line. has been detected in mFZiiA preprations prepared from FD-PMA which would support the growth of the parental cell line. Since protein kinase C (PKC) is a mediator of PMA effects, we examined this kinase. In FDC-Pl total ce!lular PKC activity was quanitated by 32-P incorporation into histone and was 24,493 CPM ("=3), However, PKC activity was undetectable in FD-PMA. Total cellular PKC activity in FD-PMA cultured in the presence of IL-3 was 7,641 CPM. In a solubilized particulate fraction, PKC activation enhanced 32-P incorporation into 17 f 48 kDa substrates in FDC-Pl but PKC-independent phosporylation of a 90 kDa not FD-PMA. Comparisons between these substrate was enhanced in FD-PMA. cell lines should provide insight into IL-3 and PMA mediated signal transduction.
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IL-lrr REGULATES INTERLEUKIN 2 GENE EXPRESSION IN MURINE THYMOMA CELLS BY A POST-INITIATION MECHANISM. M. D. Van Norstrand. S. N. Ho. M. P. Bell. R. T. Abraham and D. J. w. Dept. of Immunology, Mayo Clinic, Rochester, MN 55905. Murine thymoma subclone EL4.6.10 constitutively produces low levels of interleukin 2 (IL2) as measured by bioassay in HT-2 cells. Interleukin-lo (ILla) and phorbol myristate acetate (PMA) individually and in synergy stimulate a" increase in IL2 gene expression and protein synthesis as detected by northern analysis of total cellular mRNA and IL2 of IL2 gene bioassay, respectively. HOWeVer, meaS"reme"t transcription rates by in vitro nuclear run-on assays utilizing cDNA probes complementary to mature IL2 mRNA revealed no increase in transcription rates in response to ILla or PMA. Nuclear run-o" assays were performed with nuclei from ILla stimulated EL4.6.10 cells using dot-blot analysis with probes for either exons l-2 or exon 4. Although no increase was detected in IL2 gene transcription rates with the exe" 1-2 probe, a significant increase in transcription rate could be detected with the cDNA probe specific for exe" 4. Further, mRNA half-life exneriments using actinomvcin D to block transcription suggest that increased levels of cellular IL2 mRNA were not due to increased mature transcript stability. These data suggest that ILla regulation of IL2 gene expression in EL4.6.10 cells occurs predominantly after the initiation of transcription and may involve removal of a block to transcriptional elongation of IL2 mRNA.
MURINE CD4-CDB- THYMOCYTES ARE STIMULATED BY IL-2 TO PROLIFERATE AND ACQUIRE CD4 BY IL-2 IN VITRO IN CHEMICALLY DEFINED MEDIUM. G. W. Wood and R.M. Klein. Univ. Kansas Med. Ctr. Kansas City, K> 66103 IL-2 receptors are expressed on a high proportion of both fetal and adult CD4-CD8- thymocytes. CD4-CDS- cells are the first cells to appear in the thymus and contain precursors to all other thymocyte subsets, yet the function of constitutively During the current expressed IL-2 receptors is in doubt. study, CD4-CDE- thymocytes from inbred strains of mice were cultured in serum-free, chemically-defined medium in the presence and absence of IL-2. Some proliferation (2-8XlO3CPM) occurred in the absence of IL-2. This was increased 20-40X by Proliferation was quantified by addition of IL-2 to cultures. scintillation counting of 3H thymidine incorporation. Nuclear The total localization was confirmed by autoradiography. number of viable cells did not increase significantly during the 4 day culture period either in the presence or absence of Similar IL-2-stimulated proliferation was observed with IL-2. IL-2-stimulated thymocytes from 15-18 day-old fetal mice. receptor proliferation was completely blocked by anti-IL-2 antibody. To determine whether or not differentiation occured, viable cells were collected after 4 days of culture and phenotyped by immunocytochemistry. Over ED% of the cells had expressed CD8. These acquired CD4. Less than 5% of the cells data strongly suggest that early thymocyte proliferation lnvolves stimulation of IL-2 receptor positive CD4-CD8thymocytes by IL-2.
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MODULATION OF TUMOR ASSOCIATED MACROPHAGE ACTIVITY BY RECOMBINANT INTERFERON-GAMMA. M.J. Thomassen, T.W. Rice, H.P. Wiedemann, D.P. Meeker and M. Ahmad. Cleveland Clinic Foundation, Cleveland, OH 44195. We evaluated the cytotoxic response of tumor associated macrophages (TAMS) t" recombinant interferon-gamma (Biogen). TAMS were recovered from resected tumor tissue of 5 patients with primary lung cancer. From 4 of these patients, macrophages were also isolated from """tumor tissue. After digestion of tissue with a mixture of collagenase and DNase, the cell suspension was adhered to plastic. The resulting cell population was >90% macrophages as evidenced by morphology and nonspecific esterase staining. Macrophage yields from 7-16g of tumor tissue varied between 0.8 - 6.1 x IO6 macronhaees. From 6-16~ of """tumor tissue. 0.7 - 10.8 x lo6 macro;ha;es were obtain:d. Tumoricidal activity was assessed using 'H-thymidine labelled human tumor cells (SK-MEL-28, CRL 1718). TAMS from only l/5 tumors demonstrated spontaneous tumoricidal activity, but after exposure to interferon-gamma the TAMS from the remaining 4 tumors also demonstrated cytotoxicity against tumor targets (44.0 5 5.8, mean % cytotoxicity 2 SEM). Macrophages from """tumor tissue demonstrated similar cytotoxicity (39.5 ? 6.7) after exposure to interferon-gamma. These results suggest that the tumoricidal activity of both TAMs and """tumor tissue macrophages may be enhanced by interferon-gamma.
DIFFERENTIAL EFFECTS OF TRANSFORMING GROWTH FACTOR-B (TGF-!3) AND EPIDERMAL GROWTH FACTOR (EGF) ON THE CELL CYCLE OF CULTURED RABBIT ARTICULAR CHONDROCYTES. D.Vivieq*, E.Lebrun**, P.GalCra*+, G.Lovak* and J-P.Puiol*. Laboratoire de Biochimie du Tissu Conjonctif*, Centre de Transfusion Sanguine**, CHU C&e de Nacre, 14033 Caen Cedex, France. In presence of 2 or 10% FCS. TGF-B at 0.1. 1 and 2 “g/ml had no effect on cell proliferation after 24-h exposure. By flow cytometric analysis we demonstrated that a high proportion of cells were arrested in late S-phase at this time. In both serum concentrations, a disappearance of the blocked cells occurred at 48 h after TGF-I3 addition. At this time, an increase of cell proliferation was only seen for 10% FCS. This suggests that TGF-!3 exerta a stimulatory effect on the replication in optimal concentration of serum. We compared the effect of TGF-!3 to that of EGF. In both quiescent (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. without arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases. [3H]thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-!3 than for EGF. Although both factors induce similar increases in cell number at 48 h in 10 % FCS-containing medium, these proliferative effects were due to different actions on the cell cycle.
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ROLE OF LYMPHOCYTE FUNCTION-ASSOCIATED ANTIGEN-l (LFA-1) IN THE MIGRATION OF T CELLS TO LYMPHOCYTE CHEMOTACTIC FACTOR (LCF) AND IL-Z. M. Vachula, C.W. Smith. J. Potter. D.C. Anderson. D.E. Van Eous. Applied Sciences, Baxter Healthcare Cot-p., Round Lake, IL 60073; Baylor Cal. of Med., Houston, TX 77046; Univ. of New Mexico, Albuquerque, NM 87131. The role of LFA-1 in the chemotactic response of human T cells was examined. LCF, purified from concanavalin A stimulated peripheral blood mononuclear cells, and rIL-2 were used to stimulate T cell migration in blind well chemotaxis chambers. Chemotaxis was measured by the leading front assay using T cells prepared by E-rosetting (>90% CD3+ as determined by flow cytometry). The role of LFA-1 in migration was evaluated using antibodies to the LFA-1 a chain, CDlla (TS1/22) and I3 chain, CD18 (TS1/18). T cell chemotaxis in response to LCF and IL-2 was significantly suppressed by TS1/18 antibody and to a lesser degree by TS1/22. Neither antibody suppressed random T cell migration. Control anti-CD4 monoclonal antibody of the same IgGl isotype as the anti-LFA antibodies did not inhibit chemotaxis or random migration. Additional studies to evaluate the adherence and migration of T cells through IL-1 stimulated human umbilical vein endothelial cell monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration but failed to inhibit adherence. These studies show that LFA-1 plays a role in the migration of T cells through endothelial cells and that the R chain of LFA-1 is important in the in vitro migration of T cells in response to LCF and IL-2.
PHORBOL ESTERS CAN PERSISTENTLY REPLACE INTERLEUKIN-3 (IL-3) FOR THE GROWTH OF A IL-3-DEPENDENT CELL LINE. K. Ways, R. Riddle, L. Steelman, and J. McCubrey. East Carolina Univ. Sch. of Medicine, Greenville, NC. 27858. FDC-Pl is a" IL-3-dependent cell line which normally dies in the absence of IL-3. We have isolated a variant cell line that grows continuously in the presence of phorbol myristate acetate (PMA). This variant cell line (FD-PMA) has been maintained in culture for 10 months and optimal cell growth is achieved in PMA rather than in IL-3. LFA-1, Mac-l, and Mac-2 were readily detected on the cell surface of FD-PMA, however these antigens were not detected No lymphokine gene expression on the parental cell line. has been detected in mFZiiA preprations prepared from FD-PMA which would support the growth of the parental cell line. Since protein kinase C (PKC) is a mediator of PMA effects, we examined this kinase. In FDC-Pl total ce!lular PKC activity was quanitated by 32-P incorporation into histone and was 24,493 CPM ("=3), However, PKC activity was undetectable in FD-PMA. Total cellular PKC activity in FD-PMA cultured in the presence of IL-3 was 7,641 CPM. In a solubilized particulate fraction, PKC activation enhanced 32-P incorporation into 17 f 48 kDa substrates in FDC-Pl but PKC-independent phosporylation of a 90 kDa not FD-PMA. Comparisons between these substrate was enhanced in FD-PMA. cell lines should provide insight into IL-3 and PMA mediated signal transduction.
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IL-lrr REGULATES INTERLEUKIN 2 GENE EXPRESSION IN MURINE THYMOMA CELLS BY A POST-INITIATION MECHANISM. M. D. Van Norstrand. S. N. Ho. M. P. Bell. R. T. Abraham and D. J. w. Dept. of Immunology, Mayo Clinic, Rochester, MN 55905. Murine thymoma subclone EL4.6.10 constitutively produces low levels of interleukin 2 (IL2) as measured by bioassay in HT-2 cells. Interleukin-lo (ILla) and phorbol myristate acetate (PMA) individually and in synergy stimulate a" increase in IL2 gene expression and protein synthesis as detected by northern analysis of total cellular mRNA and IL2 of IL2 gene bioassay, respectively. HOWeVer, meaS"reme"t transcription rates by in vitro nuclear run-on assays utilizing cDNA probes complementary to mature IL2 mRNA revealed no increase in transcription rates in response to ILla or PMA. Nuclear run-o" assays were performed with nuclei from ILla stimulated EL4.6.10 cells using dot-blot analysis with probes for either exons l-2 or exon 4. Although no increase was detected in IL2 gene transcription rates with the exe" 1-2 probe, a significant increase in transcription rate could be detected with the cDNA probe specific for exe" 4. Further, mRNA half-life exneriments using actinomvcin D to block transcription suggest that increased levels of cellular IL2 mRNA were not due to increased mature transcript stability. These data suggest that ILla regulation of IL2 gene expression in EL4.6.10 cells occurs predominantly after the initiation of transcription and may involve removal of a block to transcriptional elongation of IL2 mRNA.
MURINE CD4-CDB- THYMOCYTES ARE STIMULATED BY IL-2 TO PROLIFERATE AND ACQUIRE CD4 BY IL-2 IN VITRO IN CHEMICALLY DEFINED MEDIUM. G. W. Wood and R.M. Klein. Univ. Kansas Med. Ctr. Kansas City, K> 66103 IL-2 receptors are expressed on a high proportion of both fetal and adult CD4-CD8- thymocytes. CD4-CDS- cells are the first cells to appear in the thymus and contain precursors to all other thymocyte subsets, yet the function of constitutively During the current expressed IL-2 receptors is in doubt. study, CD4-CDE- thymocytes from inbred strains of mice were cultured in serum-free, chemically-defined medium in the presence and absence of IL-2. Some proliferation (2-8XlO3CPM) occurred in the absence of IL-2. This was increased 20-40X by Proliferation was quantified by addition of IL-2 to cultures. scintillation counting of 3H thymidine incorporation. Nuclear The total localization was confirmed by autoradiography. number of viable cells did not increase significantly during the 4 day culture period either in the presence or absence of Similar IL-2-stimulated proliferation was observed with IL-2. IL-2-stimulated thymocytes from 15-18 day-old fetal mice. receptor proliferation was completely blocked by anti-IL-2 antibody. To determine whether or not differentiation occured, viable cells were collected after 4 days of culture and phenotyped by immunocytochemistry. Over ED% of the cells had expressed CD8. These acquired CD4. Less than 5% of the cells data strongly suggest that early thymocyte proliferation lnvolves stimulation of IL-2 receptor positive CD4-CD8thymocytes by IL-2.