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Macrophage migration inhibition (MMI) by human interferon (IFN) and interferon inducers
THIRD INTERNATIONAL
LYMPHOKINE
WORKSHOP
389
Migration Inhibition (MMI) by Human Interferon (IFN) and Interferon Inducers. GARY B. THURMAN AND HAROLD B. STULL, Biological Response Modifiers Program, National Cancer Institute-FCRF, Frederick, Maryland 21701.
Macrophage
Partially purified human OL,@,and y IFN are potent inhibitors of the migration of guinea pig mineral oil-induced peritoneal exudate cells (PEC). Using the agarose droplet technique, we found that purified recombinant leukocyte (a) IFN is also potent in its MM1 ability. MM1 activity has been evident at IFN concentrations as low as 10 III/ml, with y IFN appearing to have the most MM1 activity per unit of antiviral activity. IFN-inducing agents such as poly(I:C) are also effective at MM1 at concentrations that are nontoxic. These observations raise a note of caution in ascribing migration inhibitory factor (MIF) activity to antigen- or mitogen-induced supernatants that may also contain IFN or IFN-inducing agents. Supernatants from cell lines reported to make MIF must also be evaluated for IFN activity since the MIF assay does not distinguish between MIF and IFN. IFN appears to act directly on macrophages to inhibit their migration since a human macrophage-like cell line, U937, was also inhibited from migrating by IFN. The mechanism by which IFN inhibits macrophage migration is not known, but our results raise the possibility that macrophage MIF may act through the induction of IFN production, perhaps by the macrophages themselves, which in turn limits cellular migration. Requirement for y-Interferon in Activation of Macrophages for Tumor Cytotoxicity. D. N. M~NNEL AND W. FALK, Institut ftir Immunologie und Genetik, DKFZ, 69 Heidelberg, Germany.
Participation of Interferon (IFN) in macrophage activation was suggested by various investigators. y-IFN activity was always present in lymphokine preparations that were able to activate macrophages for tumor cell lysis. Semipurified IFN preparations were reported to activate macrophages and activation was inhibited by antisera against IFN. Biochemically, the two activities, macrophage-activation factor (MAF) and y-IFN, have not been separated. We found that antiviral titers always paralleled macrophage activation. A monoclonal rat antibody against murine IFN was utilized to test the requirement for yIFN in this assay. The purified antibodies neutralized IFN activity in Con A- and antigen-stimulated spleen cell supcrnatants as well as in supernatants of Con A-stimulated cloned T cells. Monoclonal antibodies coupled to CH-Sepharose 4B absorbed IFN activity in these supernatants. Unchanged Interleukin 2 titers suggested that the neutralization effect was not due to unspecific absorption by the immunosorbent. Quantitative analysis of macrophage activation by a S-hr lymphokine pulse or continuous presence of the lymphokine during the entire assay absorbed to the same degree as the IFN activity. These results show that r-IFN is essential for activation of macrophages for tumor cell lysis but the question if y-IFN and MAF are identical or if these two factors act synergistically could not yet be answered. Regulation
of Macrophage
Ia Expression.
D. I. BELLERAND E. R. UNANUE, Harvard Medical School,
Boston, Massachusetts. The expression of Ia antigens on macrophages (M4) is critical for their function as antigen-presenting cells. Since the ability of individual Mg to synthesize and express Ia is short-lived, the control mechanisms regulating these events become of major importance. Ia can be induced on peritoneal exudate Mg both in vitro and in vivo by a soluble mediator elaborated by activated T cells in the presence of antigen and Ia M$J as accessory cells. T-cell lines and hybridomas also produce this lymphokine, which appears to be a protein of about 50,000 daltons. In vitro evaluation has revealed a rapid commitment event in response to the supernatant; a 2-hr exposure is sufficient to induce the maximal level of Ia, seen after 4-7 days in culture. However, there is a requisite lag period before Ia is expressed on the M$ surface. The duration of this lag period is characteristic of the means by which the M# is elicited in vivo: activated phagocytes elicited by Listeria or thioglycollate have a brief (2-3 days) lag period, while resident M$ do not express Ia until 5-6 days after stimulation. Peptone-elicited cells are intermediate in responsiveness.There is a striking inverse correlation between the inducibility of each population and its level of the ectoenzyme 5’-nucleotidase. Moreover, at the time when resident M+ start to express membrane Ia, they have much lower levels of enzyme/rg cell protein than at the onset of culture. Induction of Ia culminates in fully restored antigen-presenting capacity. Lymphokine Induction of DR Antigen Expression on Human Neonatal and Cell Line Monocytes. M. B. SZTEIN, P. S. STEEG, D. MANN, R. STIEHM, R. M. BLASE, AND J. J. OPPENHEIM, NIDR,
NIH, Bethesda, Maryland. Normal human adult peripheral blood adherent mononuclear cells (AMC) are 75-95s DR antigen positive, while AMC from normal neonatal blood express less DR antigen (lo-25% DR+), as quantified
LYMPHOKINE
WORKSHOP
389
Migration Inhibition (MMI) by Human Interferon (IFN) and Interferon Inducers. GARY B. THURMAN AND HAROLD B. STULL, Biological Response Modifiers Program, National Cancer Institute-FCRF, Frederick, Maryland 21701.
Macrophage
Partially purified human OL,@,and y IFN are potent inhibitors of the migration of guinea pig mineral oil-induced peritoneal exudate cells (PEC). Using the agarose droplet technique, we found that purified recombinant leukocyte (a) IFN is also potent in its MM1 ability. MM1 activity has been evident at IFN concentrations as low as 10 III/ml, with y IFN appearing to have the most MM1 activity per unit of antiviral activity. IFN-inducing agents such as poly(I:C) are also effective at MM1 at concentrations that are nontoxic. These observations raise a note of caution in ascribing migration inhibitory factor (MIF) activity to antigen- or mitogen-induced supernatants that may also contain IFN or IFN-inducing agents. Supernatants from cell lines reported to make MIF must also be evaluated for IFN activity since the MIF assay does not distinguish between MIF and IFN. IFN appears to act directly on macrophages to inhibit their migration since a human macrophage-like cell line, U937, was also inhibited from migrating by IFN. The mechanism by which IFN inhibits macrophage migration is not known, but our results raise the possibility that macrophage MIF may act through the induction of IFN production, perhaps by the macrophages themselves, which in turn limits cellular migration. Requirement for y-Interferon in Activation of Macrophages for Tumor Cytotoxicity. D. N. M~NNEL AND W. FALK, Institut ftir Immunologie und Genetik, DKFZ, 69 Heidelberg, Germany.
Participation of Interferon (IFN) in macrophage activation was suggested by various investigators. y-IFN activity was always present in lymphokine preparations that were able to activate macrophages for tumor cell lysis. Semipurified IFN preparations were reported to activate macrophages and activation was inhibited by antisera against IFN. Biochemically, the two activities, macrophage-activation factor (MAF) and y-IFN, have not been separated. We found that antiviral titers always paralleled macrophage activation. A monoclonal rat antibody against murine IFN was utilized to test the requirement for yIFN in this assay. The purified antibodies neutralized IFN activity in Con A- and antigen-stimulated spleen cell supcrnatants as well as in supernatants of Con A-stimulated cloned T cells. Monoclonal antibodies coupled to CH-Sepharose 4B absorbed IFN activity in these supernatants. Unchanged Interleukin 2 titers suggested that the neutralization effect was not due to unspecific absorption by the immunosorbent. Quantitative analysis of macrophage activation by a S-hr lymphokine pulse or continuous presence of the lymphokine during the entire assay absorbed to the same degree as the IFN activity. These results show that r-IFN is essential for activation of macrophages for tumor cell lysis but the question if y-IFN and MAF are identical or if these two factors act synergistically could not yet be answered. Regulation
of Macrophage
Ia Expression.
D. I. BELLERAND E. R. UNANUE, Harvard Medical School,
Boston, Massachusetts. The expression of Ia antigens on macrophages (M4) is critical for their function as antigen-presenting cells. Since the ability of individual Mg to synthesize and express Ia is short-lived, the control mechanisms regulating these events become of major importance. Ia can be induced on peritoneal exudate Mg both in vitro and in vivo by a soluble mediator elaborated by activated T cells in the presence of antigen and Ia M$J as accessory cells. T-cell lines and hybridomas also produce this lymphokine, which appears to be a protein of about 50,000 daltons. In vitro evaluation has revealed a rapid commitment event in response to the supernatant; a 2-hr exposure is sufficient to induce the maximal level of Ia, seen after 4-7 days in culture. However, there is a requisite lag period before Ia is expressed on the M$ surface. The duration of this lag period is characteristic of the means by which the M# is elicited in vivo: activated phagocytes elicited by Listeria or thioglycollate have a brief (2-3 days) lag period, while resident M$ do not express Ia until 5-6 days after stimulation. Peptone-elicited cells are intermediate in responsiveness.There is a striking inverse correlation between the inducibility of each population and its level of the ectoenzyme 5’-nucleotidase. Moreover, at the time when resident M+ start to express membrane Ia, they have much lower levels of enzyme/rg cell protein than at the onset of culture. Induction of Ia culminates in fully restored antigen-presenting capacity. Lymphokine Induction of DR Antigen Expression on Human Neonatal and Cell Line Monocytes. M. B. SZTEIN, P. S. STEEG, D. MANN, R. STIEHM, R. M. BLASE, AND J. J. OPPENHEIM, NIDR,
NIH, Bethesda, Maryland. Normal human adult peripheral blood adherent mononuclear cells (AMC) are 75-95s DR antigen positive, while AMC from normal neonatal blood express less DR antigen (lo-25% DR+), as quantified