1
102 / SECOND INTERNATIONAL WORKSHOP ON CYTOKINES 175 EVIDENCE FOR FUNCTIONAL HETEROGENEITY IN VESICUIAR STOMATITIS VIRUS (VSV)-INDUCED HELPER T CELLS R.P.Ciavarra. Eastern Virginia Medical School, Norfolk, VA 23501. We have assessed the requirement for VSV-induced helper T (Th) cells (L3T4+,Lyt-2-) during the generation of anti- VSV CTLs. VSV memory CTLs (L3T4-,Lyt-2+) do not become effector cells when restimulated with VSV; however, VSV- specific CTLs are generated in a dose dependent fashion by the addition of VSV-induced Th cells. Llmitine dilution analysis of VSV-induced Th cells generated multi-hit curves not consistent with the view that a single limiting Th cell was responsible for conversion of memory CTL into CTL. In support of the view that more than a single type of Th cell contributed to CTL development were the observations that 1) monoclonal antibodies to either the IL-2 receptor (PC61) or IL-4 (llB11) blocked anti-VSV CTL development and 2) VSV memory CTLs did not proliferate when stimulated with VSV even in the presence of rIL-2. In contrast, the addition of a supernatant from a prototypic Th2 line (DlO) containing IL-4 but no IL-2 to cultures containing VSV memory CTLs and VSV did result in the generation of anti- VSV CTLs without the concomitant activation of NK or L4K. IL-2 alone could not reconstitute the anti-VSV CTL response but could augment CTL development in the presence of the DlO supernatant. These studies suggest that two types of antigen specific Th cells (perhaps Thl and Th2) may be required for CTL generation against VSV. 176 SYNERGISTIC EFFECTS OF TNF,LPS AND IL-la ON STIMULATING CULTURED ENDOTHELIUM PGI2 PRODUCTION. J Cohn and LC Casey. Rush-Presbyterian-St. Luke's Med Ctr, Chgo 11 60612. LPS and TNF have been implicated as mediators of septic shock. We hypothesize that low levels of LPS, TNF, and IL-la may have a synergistic effect on produc- ing endothelial cell (EC) injury. Bovine pulmonary artery EC were *g/ml), TNF (lo-ff'~~"E~~)~l,8,h~~sw~~ghl~~~o~~~~~;2~,~ combinations of the different agents. The supernatants were then assayed for 6-keto PGF la by RIA. The relative potency of the agents were LPSIl$L-la, and TNF. Doses of LPS (.Olne/ml~ or IL-la (2x10 Ml which bv themselves failed to-~ti&late PG12 &ulted in augmen&tion of the TNF dose respo se stimulation of P Likewise, doses of TNF (3xlD-A M) or IL-la (2x10- 'iI52. M) which by them- selves failed to stimulate PG12 when added separately, in combination with LPS, both potentiated the LPS dose response curve of PG12 production. These findings demonstrate a synergistic interaction of LPS, IL-la and TNF on stimulating EC production of PGI of arachidonic acid (2OuM) for 15 min. 3 The addition a ter the initial incubation with cytokines resulted in a LPS and IL-la dose dependent conversion to PG12 which was not seen with TNF or IL-lg. This data suggests that there may be more than one mechanism by which cytokines stimulate PG12. 177 INCREASE OF NEUTROPHIL FMLP AGGREGATION BY GM-CSF WITHOUT LTB4 AND TXAP RELEASE: LACK OF EFFECT OF hrlL-6. P. Conti. M. Reale. S, Fiore. R.C. Barbacane. Demosev*. Immunology Division, University of Chieti, Chieti. 66100, Italy and l Endogen, Inc., Boston, MA, 02210, U.S.A. Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein lymphokine released mainly by activated T-cells involved in immunity and inflammation. We have found that GM-CSF can prime the neutrophil response to various biological stimulants that activate them, such as leukocyte inhibitory factor &IF). We have examined the priming role of the cytokine GM-CSF on human neutrophilic aggregation stimulated by formyl-methionyf-leucyt-phenylalanine (FMLP) and its influence on the production of arachidonic acid metabolites. Aggregation of purified polymorphonuclear (PMN) cells was measured by the increase in light transmission in a standard aggregometer (four channels). LTB4 and TX& two arachidonic acid products that mediate inflammatory responses, were measured by RIA from the PMN supernatants after treatment with GM-CSF or IL-6 We found that GM-CSF, 11-6, IL-1 and TNF when used alone do not cause any significant increase in light transmission of the PMN cell cultures, whereas when FMLP was added after GM-CSF exposure. a significant increase in aggregation was achieved compared with FMLP alone. This priming effect on aggregation was not obtained with IL-l. TNF or 11-6. GM-CSF and IL-6 alone failed to stimulate LTB4 or TxA2 production by PMN. These findings provide new evidence suggesting that GM-CSF facilitates the action of FMLP on the adhesion- dependent cellular function of the inflammatory response. These data support the hypothesis that GM-CSF acts as an aggregating co-factor and as a direct activator of human PMN function (such as ADCC and phagocytosis). Thus, GM-CSF may, by itself, play a key role in neutrophil immunoregulation. 178 LIF POTENTIATES MACROPHAGE AGGREGATION INDUCED BY CALCIUM IONOPHORE (A23187) AND POTENTLY GENERATES LTB4 AND TxA2 RELEASE BY HUMAN MACROPHAGES. P. Conti. M. Reale. S. Fiore. R.C. Barbacane. M.R. Panara. M.Bonarazio. L. Borish* J.M. Castracane* and R.A. Dempsey”. Immunology Division, University of Chieti, Chieti, 66100, Italy and Endogen, Inc., Boston, MA, 02210. U.S.A. Leukocyte inhibitory factor (LIF) is a lymphokine we have purified 60,000 fold to apparent homogeneity and has an apparent M.W. of 56,000. LIF is a glycoprotein that was initially defined by its ability to inhibit the random movement or directed migration of polymorphonuclear (PMN) leukocytes. Recently we have demonstrated that LIF is also a very potent neutrophil activating factor (NAF) and potentiates several neutrophil responses to form@methionyl-leucyi-phenytalanine (FMLP) such as chemotaxis, superoxide (SO) generation and PMN degranulation, possibly by increasing the number of available membrane receptors. We studied the ability of LIF to potentiate macrophage (MO) aggregation before exposure to calcium ionophore A23187 and fis capacity to directly stimulate the two proaggregation arachidonic acid metabolites LTB4 and TxA2. MO aggregation was quantitated by measuring the increase in light transmission using a four channel aggregometer. LTB4 and TxAZ (detected as TxBP) were measured by specific RIAs. We found that pretreatment of MO with LIF significantly potentiates the aggregation response to calcium ionophore, however LIF itself did not provoke any significant aggregation. LIF is a potent stimulator of the two proinflammatory AA products LTB4 and TxA2. The direct stimulation of MO by LIF of the lipoxygenase and cyclooxygenase products LTB4 and TxAZ provides new evidence suggesting a greater role for this lymphokine in the granulocyte- macrophage group of leukocytes which have a major influence on inflammation and host defence. 179 CYTOKINE PRODUCTION IS A FUNCTION OF MURINE UTERINE GRANULATED METRIAL GLAND (GMG) CELLS. B. Anne Croy, Dan Stinchcomb, Nicholas M. Gough and Larry J. Guilbert. Dept. Biomedical fi ON, Can., NlG ZWl, Sciences, ' ' Synerqen. Boulder, CO, 80301, Walter & Eliza Hall Institute of Mkdical Research,.Melbourne,.Victoria 3050, Australia and Dept. Immunology, University of Alberta, Edmonton, AB, Can., T6G 2H7. GMG cells are bone marrow-derived uterine cells with a large granulated lymphoid phenotype that, in rodents, differentiate at implantation sites and migrate into the mature placenta. Their functions are unknown. While GMG cells are unlikely to be T, B or NK cells, interest in the influence of lympho-hematopoietic cytokines on development has led us to determine the profile of cytokine productions by GMG cells in supernatants recovered from pure populations of GMG cells that migrate from explant cultures. In vitro bioassays were used to demonstrate the absence of IL-Z, GM-CSF, IL-3, IL-4, TNF, TGF-8, IFNy and the presence of IL-l. High levels of CSF-1 were detected using a specific radioreceptor assay since supernatants killed the CSF-1 dependent macrophage cell line 5. 10.14. mRNA encoding LIF was also demonstrated in this cell population. Thus, one activity of GMG cells is cytokine production. The unique association of these cells with pregnancy and pseudopregnancy suggests that certain cytokines may be essential for embryonic success and may be regulated by the steroid hormones progesterone and estrogen. Supported by awards from NSERC and OMAF. 180 MACROPHAGE COLONY STIMULATING FACTOR ACTIVATES KUPPFER CELLS TO A CYTOTOXIC STATE. S. Curley H Roh E. Kleinerman, J. Klostergaard. M.D.Anderson Cancer'Center,'Houston TX 7703c Hacrophage colony stimulating factor (NCSF) has been shown to activate peripheral blood monocytes in vitro. We have investigated the ability -- of recombinant MCSP lo activate murine Kupffer cells (KC). KC were isolated from Balb/C mice and treated in vitro with either endotoxin-free MCSF (1000 U/ml), gamma-interferon (y-IFN, 100 U/ml) or MCSF followed by y-IFN. Cell-free supernatants were collected after 24-72 hours and added.to a murin with '25 olon adenocarcinoma cell line (HCA26) labeled I-iododeoxyuridine. Cytotoxicity was expressed as radiolabel release at 72 hours. MCSF activated KC supernatants produced cytotoxicity in the HCA26 target cells (25%) which was maximal 24 hours after activation. y-IFN activated KC SupernaCant cytotoxicity (36%) was greater than the MCSF activated group. There was no synergistic effect on cytotoxicity observed combining HCSF and y-IFN (37%). KC supernatant cytotoxicity against MCA26 targets has been shown to be tumor necrosis factor (TNF) dependent. KC activated by NCSF or V-IFN oroduced TNF as measured bv L929 cvtolvsis assav. These re&ts demonstrate that (1) H&F can acti&te mu&e KC to a cytotoxic state and (2) MCSF and y-IFN do not appear to act synergistically in activating KC. MCSF may have a role in viva in activating tissue macrophages CytotoZcityagainst tumor cells. to produce

Cytokine production is a function of murine uterine granulated metrial gland (GMG) cells

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102 / SECOND INTERNATIONAL WORKSHOP ON CYTOKINES

175

EVIDENCE FOR FUNCTIONAL HETEROGENEITY IN VESICUIAR STOMATITIS VIRUS (VSV)-INDUCED HELPER T CELLS R.P.Ciavarra. Eastern Virginia Medical School, Norfolk, VA 23501.

We have assessed the requirement for VSV-induced helper T (Th) cells (L3T4+,Lyt-2-) during the generation of anti- VSV CTLs. VSV memory CTLs (L3T4-,Lyt-2+) do not become effector cells when restimulated with VSV; however, VSV- specific CTLs are generated in a dose dependent fashion by the addition of VSV-induced Th cells. Llmitine dilution analysis of VSV-induced Th cells generated multi-hit curves not consistent with the view that a single limiting Th cell was responsible for conversion of memory CTL into CTL. In support of the view that more than a single type of Th cell contributed to CTL development were the observations that 1) monoclonal antibodies to either the IL-2 receptor (PC61) or IL-4 (llB11) blocked anti-VSV CTL development and 2) VSV memory CTLs did not proliferate when stimulated with VSV even in the presence of rIL-2. In contrast, the addition of a supernatant from a prototypic Th2 line (DlO) containing IL-4 but no IL-2 to cultures containing VSV memory CTLs and VSV did result in the generation of anti- VSV CTLs without the concomitant activation of NK or L4K. IL-2 alone could not reconstitute the anti-VSV CTL response but could augment CTL development in the presence of the DlO supernatant. These studies suggest that two types of antigen specific Th cells (perhaps Thl and Th2) may be required for CTL generation against VSV.

176

SYNERGISTIC EFFECTS OF TNF,LPS AND IL-la ON STIMULATING CULTURED ENDOTHELIUM PGI2 PRODUCTION. J Cohn and LC Casey. Rush-Presbyterian-St. Luke's Med Ctr, Chgo 11 60612.

LPS and TNF have been implicated as mediators of septic shock. We hypothesize that low levels of LPS, TNF, and IL-la may have a synergistic effect on produc- ing endothelial cell (EC) injury. Bovine pulmonary artery EC were *g/ml),

TNF (lo-ff'~~"E~~)~l,8,h~~sw~~ghl~~~o~~~~~;2~,~

combinations of the different agents. The supernatants were then assayed for 6-keto PGF la by RIA. The relative potency of the agents were LPSIl$L-la, and TNF. Doses of LPS (.Olne/ml~ or IL-la (2x10 Ml which bv themselves failed to-~ti&late PG12 &ulted in augmen&tion of the TNF dose respo se stimulation of P Likewise, doses of TNF (3xlD-A M) or IL-la (2x10- 'iI52. M) which by them- selves failed to stimulate PG12 when added separately, in combination with LPS, both potentiated the LPS dose response curve of PG12 production. These findings demonstrate a synergistic interaction of LPS, IL-la and TNF on stimulating EC production of PGI of arachidonic acid (2OuM) for 15 min. 3

The addition a ter the initial

incubation with cytokines resulted in a LPS and IL-la dose dependent conversion to PG12 which was not seen with TNF or IL-lg. This data suggests that there may be more than one mechanism by which cytokines stimulate PG12.

177

INCREASE OF NEUTROPHIL FMLP AGGREGATION BY GM-CSF WITHOUT LTB4 AND TXAP RELEASE: LACK OF EFFECT OF hrlL-6. P. Conti. M. Reale. S, Fiore. R.C. Barbacane. Demosev*. Immunology Division, University of Chieti, Chieti. 66100, Italy and l Endogen, Inc., Boston, MA, 02210, U.S.A.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein lymphokine released mainly by activated T-cells involved in immunity and inflammation. We have found that GM-CSF can prime the neutrophil response to various biological stimulants that activate them, such as leukocyte inhibitory factor &IF). We have examined the priming role of the cytokine GM-CSF on human neutrophilic aggregation stimulated by formyl-methionyf-leucyt-phenylalanine (FMLP) and its influence on the production of arachidonic acid metabolites. Aggregation of purified polymorphonuclear (PMN) cells was measured by the increase in light transmission in a standard aggregometer (four channels). LTB4 and TX& two arachidonic acid products that mediate inflammatory responses, were measured by RIA from the PMN supernatants after treatment with GM-CSF or IL-6 We found that GM-CSF, 11-6, IL-1 and TNF when used alone do not cause any significant increase in light transmission of the PMN cell cultures, whereas when FMLP was added after GM-CSF exposure. a significant increase in aggregation was achieved compared with FMLP alone. This priming effect on aggregation was not obtained with IL-l. TNF or 11-6. GM-CSF and IL-6 alone failed to stimulate LTB4 or TxA2 production by PMN. These findings provide new evidence suggesting that GM-CSF facilitates the action of FMLP on the adhesion- dependent cellular function of the inflammatory response. These data support the hypothesis that GM-CSF acts as an aggregating co-factor and as a direct activator of human PMN function (such as ADCC and phagocytosis). Thus, GM-CSF may, by itself, play a key role in neutrophil immunoregulation.

178

LIF POTENTIATES MACROPHAGE AGGREGATION INDUCED BY CALCIUM IONOPHORE (A23187) AND POTENTLY GENERATES LTB4 AND TxA2 RELEASE BY HUMAN MACROPHAGES. P. Conti. M. Reale. S. Fiore. R.C. Barbacane. M.R. Panara. M.Bonarazio. L. Borish* J.M. Castracane* and R.A. Dempsey”. Immunology Division, University of Chieti, Chieti, 66100, Italy and Endogen, Inc., Boston, MA, 02210. U.S.A.

Leukocyte inhibitory factor (LIF) is a lymphokine we have purified 60,000 fold to apparent homogeneity and has an apparent M.W. of 56,000. LIF is a glycoprotein that was initially defined by its ability to inhibit the random movement or directed migration of polymorphonuclear (PMN) leukocytes. Recently we have demonstrated that LIF is also a very potent neutrophil activating factor (NAF) and potentiates several neutrophil responses to form@methionyl-leucyi-phenytalanine (FMLP) such as chemotaxis, superoxide (SO) generation and PMN degranulation, possibly by increasing the number of available membrane receptors. We studied the ability of LIF to potentiate macrophage (MO) aggregation before exposure to calcium ionophore A23187 and fis capacity to directly stimulate the two proaggregation arachidonic acid metabolites LTB4 and TxA2. MO aggregation was quantitated by measuring the increase in light transmission using a four channel aggregometer. LTB4 and TxAZ (detected as TxBP) were measured by specific RIAs. We found that pretreatment of MO with LIF significantly potentiates the aggregation response to calcium ionophore, however LIF itself did not provoke any significant aggregation. LIF is a potent stimulator of the two proinflammatory AA products LTB4 and TxA2. The direct stimulation of MO by LIF of the lipoxygenase and cyclooxygenase products LTB4 and TxAZ provides new evidence suggesting a greater role for this lymphokine in the granulocyte- macrophage group of leukocytes which have a major influence on inflammation and host defence.

179

CYTOKINE PRODUCTION IS A FUNCTION OF MURINE UTERINE GRANULATED METRIAL GLAND (GMG) CELLS. B. Anne Croy, Dan Stinchcomb, Nicholas M. Gough and Larry J. Guilbert. Dept. Biomedical fi ON, Can., NlG ZWl, Sciences, ' ' Synerqen. Boulder, CO, 80301, Walter & Eliza Hall Institute of Mkdical Research,.Melbourne,.Victoria 3050, Australia and Dept. Immunology, University of Alberta, Edmonton, AB, Can., T6G 2H7.

GMG cells are bone marrow-derived uterine cells with a large granulated lymphoid phenotype that, in rodents, differentiate at implantation sites and migrate into the mature placenta. Their functions are unknown. While GMG cells are unlikely to be T, B or NK cells, interest in the influence of lympho-hematopoietic cytokines on development has led us to determine the profile of cytokine productions by GMG cells in supernatants recovered from pure populations of GMG cells that migrate from explant cultures. In vitro bioassays were used to demonstrate the absence of IL-Z, GM-CSF, IL-3, IL-4, TNF, TGF-8, IFNy and the presence of IL-l. High levels of CSF-1 were detected using a specific radioreceptor assay since supernatants killed the CSF-1 dependent macrophage cell line 5. 10.14. mRNA encoding LIF was also demonstrated in this cell population. Thus, one activity of GMG cells is cytokine production. The unique association of these cells with pregnancy and pseudopregnancy suggests that certain cytokines may be essential for embryonic success and may be regulated by the steroid hormones progesterone and estrogen.

Supported by awards from NSERC and OMAF.

180

MACROPHAGE COLONY STIMULATING FACTOR ACTIVATES KUPPFER CELLS TO A CYTOTOXIC STATE. S. Curley H Roh E. Kleinerman, J. Klostergaard. M.D.Anderson Cancer'Center,'Houston TX 7703c

Hacrophage colony stimulating factor (NCSF) has been shown to activate peripheral blood monocytes in vitro. We have investigated the ability

-- of recombinant MCSP lo activate

murine Kupffer cells (KC). KC were isolated from Balb/C mice and treated in vitro with either endotoxin-free MCSF (1000 U/ml), gamma-interferon (y-IFN, 100 U/ml) or MCSF followed by y-IFN. Cell-free supernatants were collected after 24-72 hours and added.to a murin

with '25 olon adenocarcinoma

cell line (HCA26) labeled I-iododeoxyuridine. Cytotoxicity was expressed as radiolabel release at 72 hours. MCSF activated KC supernatants produced cytotoxicity in the HCA26 target cells (25%) which was maximal 24 hours after activation. y-IFN activated KC SupernaCant cytotoxicity (36%) was greater than the MCSF activated group. There was no synergistic effect on cytotoxicity observed combining HCSF and y-IFN (37%). KC supernatant cytotoxicity against MCA26 targets has been shown to be tumor necrosis factor (TNF) dependent. KC activated by NCSF or V-IFN oroduced TNF as measured bv L929 cvtolvsis assav. These re&ts demonstrate that (1) H&F can acti&te mu&e KC to a cytotoxic state and (2) MCSF and y-IFN do not appear to act synergistically in activating KC. MCSF may have a role in viva in activating tissue macrophages CytotoZcityagainst tumor cells.

to produce