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Thymosins α1 and β4 potentiate the antigen presenting capacity of macrophages
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POSSIBLE MECHANISM(S) RESPONSIBI.~ FOR THE CONTROL OF CELL PROLIFERATION BY HEPARAN SULFAlL. I.E. Silber, J.M. Walenga, J. Fareed, and E.J. Kovacs Departments of Anatomy and Pathology, Loyola Univ. ,Mn.. good, IL 60153. In this study we examined the regulatory effects of heparan sulfate (HS) on the growth of murine smooth muscle cells (SMC) and fihroblasts. Proliferation wes measured by monitoring cell growth after 4 days of incubation with medium supplemented with 10% f*!tal bovine serum (PBS) in the presence or absence of HS (lo-.:20 ug/ml). The addition of HS caused a dose dependent inh:bition of both SMC and fibroblast growth compared to cells incubated with medium The inhibitor of SMC ai,d fibroblast growth reached sl0ne. 100% and 70% respectively at 130uglml of HS. The continuous presence of HS was not needed .o cause growth inhibition, suggest in that the inhibitory effect of HS on cell proliferation may not merely be due to interaction of HS with growth factors. We cannot rule out the possibility that HS plays a role in the regulation of cell surface molecules involved in substrate attachment far growth factor signal transduction. These results are also directly relevant to the control of the aberrant wound healing which is the hallmark of a variety of diseases including atherosclerosis and pulmonary Ftbrosis. Supported by the Office of Naval Research.
PRODUCTION CELLS AND
OF INTERLEUKIN-6 BY RAT ANTERIOR PITUITARY HEMIPITUITARY TISSUE I/U VITRO. B.C. Soanaelo.
R.M. MacLeod and PC. Isakson. Depts. Medicine and Pharmacology, Univ. of Virginia Health Sciences Center, Charlottesville, VA 22908. We recently reported that picomolar concentrations of interleukin-6 (IL-6) can stimulate the release of prolactin, growth hormone and luteinizing hormone from anterior pituitary ceils in vitro. Since normal serum levels of IL-6 are < 10 pg/ml, we hypothesized that the anterior pituitary produces this cytokine, which functions in a paracrine and/or autocrine manner to regulate hormone secretion. Conditioned media from cultured anterior pituitary cells and hemipituitary tissue were assayed for their IL-6 contents using the IL-6 dependent 7TDl cell line. Spontaneous release of IL-6 from cuitured cells ranged from 200-1900 U/ml/4 h and was linear for 8 h, reaching levels of 2DOO-4000 U/ml (80-160 PM). Both LPS (l-1000 rig/ml) and PMA (IO-100 nM) caused a 3- to 4-fold enhancement of IL-6 production. Following a 2 h lag period, hemipituitary tissue also released IL-6 (2000-5000 U/ml/4 h). LPS (10 MS/ml) stimulated IL-6 release from these glands (2- to 4-fold increase at 4 h). IL-la and TNFa were found to have no effect upon IL-6 release from anterior pituitary cells. These data suggest that anterior pituitary cells may produce IL-6 in situ, where it may function as an intrapituitary regulator of hormone secretion. [Supported by grants from the NCI (CA-07535) and Fidia Pharmaceutical Corp. to R.M.M. and a new initiative grant from the University of Virginia DERC (AM 22125) to P.C.I.]
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KILLING OF PLASMODIUM CYNOMOLGI BY SECRETORY PRODUCTS OF MACROPHAGES ACTIVATED BY LYMPHOKINES AND RECOMBINANT HUMAN INTERFERON$.P.P.Singh and G.P.Dutta. Central Drug Res. Instt. Lucknow001, India. Activated macrophages (Ma, which secrete various substances, are vital to host-defence in malaria. We report that P.cynomolgi induced lymphokines (LK) and recombinant human interferon-r (~HuIFN-63 can activate monkey monocytederived Mbs in -Ivitro. to secrete soluble products that can kill intraerythrocytic stages of P.cynomolgi, --in vitro. LK were from antiqen-pulsed sDleen calsemonkeVs chronically infected with P,cyno&lgi. For in vitrd killing, P.cynomolgi infected monkey erythrocytes Ti;E) were incubatedwithulture supernatants (CS) of M#s activated with LK or rHuIFN-Y (72 h exposure), for 24-48 h; the parasite viability was assessed by inoculating treated IE into a normal monkey. Monkeys infected with treated IE became patent nearly 12-16 days after the controls. The delay in the onset of patency was directly proportional to the duration of the exposure of :E ot CS of activated M@.; 48 h exposure induced maximum killing. The secretory products of activated M&, present in thecrpartially killed the IE and thus reduced the size of the infective inoculum, resulting in the delay of patency.
RESTORATION OF NONMAL P-ADRENOCEPTOR CONCENTRATIONS IN A549 LUNG ADENOCANCINONA CELLS BY LEUKOCYTE PROTEIN FACTORS AND NBCONBINANT INTENLEUKIN-lo (IL-la). A. Szentivanvi, S. Reiner, M.E. Schwartz, 0. Heim, J. Szentivanyi and S. Robicsek. University of South Florida, Tampa, FL 33612 and All Children's Hospital, St. Petersburg, FL 33701. Stern and Kunos (J. Biol. Chem., 1988) demonstrated that co-culturing of IM9 human lymphocytes with A549 human lung adenocarcinoma cells results in an increase in P-adrenoceptor concentrations in the lung cells. This receptor upregulating activity was due to the release of lymphocytic protein factors including IL-1 and involved protein synthesis. Our group has demonstrated that &adrenergic dysfunction associated with a decrease in p-receptors is a pathogenic feature of bronchial asthma. Prompted by the aforementioned study, A549 cells were downregulated by in vitro exposure to isoproterenol and co-cultured with isolated leukocytes from normal volunteers or rIL-la. We found that both padrenergic responsiveness and P-adrenoceptor concentrations of the A549 lung cells were restored to normal values. Conversely, we find in preliminary experiments that replacement of normal lymphocytes by those obtained from patients with status asthmaticus shows a significant reduction in the upregulating activity.
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GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) AUGMENTS HUMAN MONOCYTE EFFECTOR AND ACCESSORY CELL FUNCTION. PD Smith. EN Janoff and SM Wahl. NIH, Bethesda, MD 20892. We investigated the effect of recombinant human GM-CSF on human monocyte effector and accessory cell activities. To assay effector activity, purified monocytes were incubated for 14 hrs with GM-CSF (5-500 U/ml) and then assayed far secretion of 0; and cytotoxicity for C. albicans. GM-CSFtreated monocy;es prdduced enha-riced levels of 0; and exhibited greater fungicidal activity than control monocytes. To test for accessory cell function, GM-CSFtreated and control monocytes were pulsed with either uhvtohemaeelutinin (PHA). Dokeweed mitoaen (PWM). Bt;eptoly& 0 (SLOj &&&us toxoid TTT) and cultured with autologous lymphocytes in a proliferation assay. Compared to control monocytes, GM-CSF-treated monocytes caused significantly greater increases in mitogenand antigen-stimulated lymphocyte proliferation and supported increased PWM-induced IgG and I.gM production. To define the mechanism of GM-CSF s.ug&ntation of accessory cell activity, surface HLA-DR and interleukin 1 (IL-l) secretion were monitored following GM-CSF treatment. Although GM-CSF it did not upregulate HLA-DR gene enhanced HLA-DR levels, SXpKeSSiOll. Incontrast, GM-CSF induced IL-1 secretion as well as IL-1 gene expression. We conclude that GM-CSF prOmOtes host defense by stimulating monocyte oxidative metabolism and cytotoxicity, and by enhancing monocyte accessory cell function.
THYMOSINS
PRESENTING E. Tzehoval
a
AND
&ACITY and
POTENTIATE /3 0% MACROPEAGES.
TEE M.B.
ANTIGEN Sztein,
/ SECOND
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
271
274
POSSIBLE MECHANISM(S) RESPONSIBI.~ FOR THE CONTROL OF CELL PROLIFERATION BY HEPARAN SULFAlL. I.E. Silber, J.M. Walenga, J. Fareed, and E.J. Kovacs Departments of Anatomy and Pathology, Loyola Univ. ,Mn.. good, IL 60153. In this study we examined the regulatory effects of heparan sulfate (HS) on the growth of murine smooth muscle cells (SMC) and fihroblasts. Proliferation wes measured by monitoring cell growth after 4 days of incubation with medium supplemented with 10% f*!tal bovine serum (PBS) in the presence or absence of HS (lo-.:20 ug/ml). The addition of HS caused a dose dependent inh:bition of both SMC and fibroblast growth compared to cells incubated with medium The inhibitor of SMC ai,d fibroblast growth reached sl0ne. 100% and 70% respectively at 130uglml of HS. The continuous presence of HS was not needed .o cause growth inhibition, suggest in that the inhibitory effect of HS on cell proliferation may not merely be due to interaction of HS with growth factors. We cannot rule out the possibility that HS plays a role in the regulation of cell surface molecules involved in substrate attachment far growth factor signal transduction. These results are also directly relevant to the control of the aberrant wound healing which is the hallmark of a variety of diseases including atherosclerosis and pulmonary Ftbrosis. Supported by the Office of Naval Research.
PRODUCTION CELLS AND
OF INTERLEUKIN-6 BY RAT ANTERIOR PITUITARY HEMIPITUITARY TISSUE I/U VITRO. B.C. Soanaelo.
R.M. MacLeod and PC. Isakson. Depts. Medicine and Pharmacology, Univ. of Virginia Health Sciences Center, Charlottesville, VA 22908. We recently reported that picomolar concentrations of interleukin-6 (IL-6) can stimulate the release of prolactin, growth hormone and luteinizing hormone from anterior pituitary ceils in vitro. Since normal serum levels of IL-6 are < 10 pg/ml, we hypothesized that the anterior pituitary produces this cytokine, which functions in a paracrine and/or autocrine manner to regulate hormone secretion. Conditioned media from cultured anterior pituitary cells and hemipituitary tissue were assayed for their IL-6 contents using the IL-6 dependent 7TDl cell line. Spontaneous release of IL-6 from cuitured cells ranged from 200-1900 U/ml/4 h and was linear for 8 h, reaching levels of 2DOO-4000 U/ml (80-160 PM). Both LPS (l-1000 rig/ml) and PMA (IO-100 nM) caused a 3- to 4-fold enhancement of IL-6 production. Following a 2 h lag period, hemipituitary tissue also released IL-6 (2000-5000 U/ml/4 h). LPS (10 MS/ml) stimulated IL-6 release from these glands (2- to 4-fold increase at 4 h). IL-la and TNFa were found to have no effect upon IL-6 release from anterior pituitary cells. These data suggest that anterior pituitary cells may produce IL-6 in situ, where it may function as an intrapituitary regulator of hormone secretion. [Supported by grants from the NCI (CA-07535) and Fidia Pharmaceutical Corp. to R.M.M. and a new initiative grant from the University of Virginia DERC (AM 22125) to P.C.I.]
272
275
KILLING OF PLASMODIUM CYNOMOLGI BY SECRETORY PRODUCTS OF MACROPHAGES ACTIVATED BY LYMPHOKINES AND RECOMBINANT HUMAN INTERFERON$.P.P.Singh and G.P.Dutta. Central Drug Res. Instt. Lucknow001, India. Activated macrophages (Ma, which secrete various substances, are vital to host-defence in malaria. We report that P.cynomolgi induced lymphokines (LK) and recombinant human interferon-r (~HuIFN-63 can activate monkey monocytederived Mbs in -Ivitro. to secrete soluble products that can kill intraerythrocytic stages of P.cynomolgi, --in vitro. LK were from antiqen-pulsed sDleen calsemonkeVs chronically infected with P,cyno&lgi. For in vitrd killing, P.cynomolgi infected monkey erythrocytes Ti;E) were incubatedwithulture supernatants (CS) of M#s activated with LK or rHuIFN-Y (72 h exposure), for 24-48 h; the parasite viability was assessed by inoculating treated IE into a normal monkey. Monkeys infected with treated IE became patent nearly 12-16 days after the controls. The delay in the onset of patency was directly proportional to the duration of the exposure of :E ot CS of activated M@.; 48 h exposure induced maximum killing. The secretory products of activated M&, present in thecrpartially killed the IE and thus reduced the size of the infective inoculum, resulting in the delay of patency.
RESTORATION OF NONMAL P-ADRENOCEPTOR CONCENTRATIONS IN A549 LUNG ADENOCANCINONA CELLS BY LEUKOCYTE PROTEIN FACTORS AND NBCONBINANT INTENLEUKIN-lo (IL-la). A. Szentivanvi, S. Reiner, M.E. Schwartz, 0. Heim, J. Szentivanyi and S. Robicsek. University of South Florida, Tampa, FL 33612 and All Children's Hospital, St. Petersburg, FL 33701. Stern and Kunos (J. Biol. Chem., 1988) demonstrated that co-culturing of IM9 human lymphocytes with A549 human lung adenocarcinoma cells results in an increase in P-adrenoceptor concentrations in the lung cells. This receptor upregulating activity was due to the release of lymphocytic protein factors including IL-1 and involved protein synthesis. Our group has demonstrated that &adrenergic dysfunction associated with a decrease in p-receptors is a pathogenic feature of bronchial asthma. Prompted by the aforementioned study, A549 cells were downregulated by in vitro exposure to isoproterenol and co-cultured with isolated leukocytes from normal volunteers or rIL-la. We found that both padrenergic responsiveness and P-adrenoceptor concentrations of the A549 lung cells were restored to normal values. Conversely, we find in preliminary experiments that replacement of normal lymphocytes by those obtained from patients with status asthmaticus shows a significant reduction in the upregulating activity.
213
216
GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) AUGMENTS HUMAN MONOCYTE EFFECTOR AND ACCESSORY CELL FUNCTION. PD Smith. EN Janoff and SM Wahl. NIH, Bethesda, MD 20892. We investigated the effect of recombinant human GM-CSF on human monocyte effector and accessory cell activities. To assay effector activity, purified monocytes were incubated for 14 hrs with GM-CSF (5-500 U/ml) and then assayed far secretion of 0; and cytotoxicity for C. albicans. GM-CSFtreated monocy;es prdduced enha-riced levels of 0; and exhibited greater fungicidal activity than control monocytes. To test for accessory cell function, GM-CSFtreated and control monocytes were pulsed with either uhvtohemaeelutinin (PHA). Dokeweed mitoaen (PWM). Bt;eptoly& 0 (SLOj &&&us toxoid TTT) and cultured with autologous lymphocytes in a proliferation assay. Compared to control monocytes, GM-CSF-treated monocytes caused significantly greater increases in mitogenand antigen-stimulated lymphocyte proliferation and supported increased PWM-induced IgG and I.gM production. To define the mechanism of GM-CSF s.ug&ntation of accessory cell activity, surface HLA-DR and interleukin 1 (IL-l) secretion were monitored following GM-CSF treatment. Although GM-CSF it did not upregulate HLA-DR gene enhanced HLA-DR levels, SXpKeSSiOll. Incontrast, GM-CSF induced IL-1 secretion as well as IL-1 gene expression. We conclude that GM-CSF prOmOtes host defense by stimulating monocyte oxidative metabolism and cytotoxicity, and by enhancing monocyte accessory cell function.
THYMOSINS
PRESENTING E. Tzehoval
a
AND
&ACITY and
POTENTIATE /3 0% MACROPEAGES.
TEE M.B.
ANTIGEN Sztein,