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Alpha-macroglobulins enhance PDGF-stimulated proliferation of lung fibroblasts in vitro
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INFECTION WITH MYCOBACTERIUM AVIUM COMPLEX (MAC) STIMULATES FIBROBLASTS (FI) TO PRODUCE GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR AND INTERFERON-BETA. L. Bermudez. Kuzell Institute, San Francisco, CA 94115. MAC is the most common bacterial agent isolated from AIDS patients. In AIDS, MAC is associated with disseminated disease, and is found invading and replicating within macrophages and monocytes. Early in the infection, MAC could invade Fi and endothelial cells. Murine (L929) Fi cell line was infected with a serotype 1 of MAC. The duplication time intracellularly was 40 hours. Progressive intracellular growth ultimately resulted in the destruction of infected cells. Supernatant obtained from infected Fi contained IFN)3 and GM-CSF [50+12 ng and 26090 pg. respectively by 18 hours). IFN)3 could be detected by 3 hours after infection, while GM-CSF was first detected by 6 hours. Treatment of infected Fi with TNF, (10 to 103 U/ml) and GM-CSF (10 to 102 U/ml) was associated with inhibition of the intracellular growth of MAC (62+7% and 60+4%. respectively). Release of cytokines from fibroblasts infected with MAC can subsequently activate macrophages and NK cells, initiating the host defense against the infection.
ENHANCED IN VITRO RECRUITERNT OF LAK ACTIVITY IN IN VIVO PRIMED LWOCYTES. R.L.H. Bolhuis, G. Stoter and C.Lamers. Daniel Den Hoed Cancer Center, Rotterdam, lhe Netherlands. NK and LAK activities of lymfocytes from patients receiving adoptive cellular immunotherapy: in vivo IL-2 activated lymfocytes show enhanced recruitement 3 ljXc activity upon in vitro 11-Z activation, but no dependency on 11-2. Cryopreservation only slightly lowers NK- and LAK activities of in vitro 11-2 activated MNC obtained from in vivo 11-2 primed cancer patients. NK and LAK activities were decreased following cryopreservation but activity was reconstituted by resting the MNC for eighteen hours. Presence of IL-2 during the cytolitic assay increased in vivo induced but not in vitro induced LAK activity. Kinetic analysis of the 11-Z enhanced cytolysis demonstrated that the enhancement of lytic activities recycled from recruitement of LAK activity but not from an intrinsic dependency of lymfocytes on 11-2. Conclusions: l-Addition of 11-2 during cytolysis for imnuneparameter monitoring may lead to an overestimate of lytic activity. e-The data suggest that prolonged in vivo administration of low-dose 11-2 will maintain maximum levels of in vivo LAK activity in the patient.
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MACROPHAGE-COLONY STIMULATING FACTOR (M-CSF) PRIMES HUMAN MACROPHAGES (MO) FOR ACTIVATION BY OTHER CYTOKINES. L. Bermudez. Kuzell Institute, San Francisco. CA 94115. MO stimulation is a chief step in the host defense against intracellular infectious microorganisms. We examined the sequential stimulation of human monocytederived MO using Mycobacterium auium (MAC) serotype 1 as a model of intracellular Infection. MO cultured in vitro for 7 days were infected with MAC and treated with recombinant IFNy, M-CSF, GM-CSF. TNF and IL-4, using different doses and sequences of stimulation. After four days, monolayers were lysed and the number of viable intracellular bacilli were quantitated after plating. M-CSF showed to be an excellent primer for further activation with lower concentrations of GM-CSF and TNF [85-o% and 88?4% of intracellular killing of MAC, respectively, compared to 26+4% and 29*7% of each alone at the same concentration (1 U/ml)). When used in inverse sequencing (TNF or GM-CSF, followed by M-CSF), the resultant intracellular killing was significantly smaller (1726% for GM-CSF/M-CSF and 3ti4% for TNF/M-CSF). These results suggest that combined treatment of human MO with cytokines may reproduce the physiologic sequence of cell activation.
AL~ROGU)BIIlINSBICWYCE~-~FUAW~LIFERRTIONOF~ FIWBLAm IN VITlXl. J.C. Banner, hi. Hoffnan, A. B&e& A. Osunic-Vat-gas tiA.R.Brod.Lab.o'fPul Pthob'l Nat1 1st fEni Hlth mg. PK., NC. ZG. ofk&l., ihi:. ofoK, Gi Hill; NC. 3??7K-? Alpha-nacrcglcbulins (a-k) secreted byalveolartnacrophages areplateletderived q&h factcr (KHZ) binding pnteins that cq&e with cell-surface recepks cn rat lung fib&lasts fcr KGF (ker. J. Resp. Cell bl. Biol., In Press, 1959). Since au-e than 50% of the FDGF secreted by these lug nocro&qesiscarplexeitoa+i,amiPDGFisaccnpetmcefactati fikcblast gru&h, we p3stulatezi that a-& mxlulate the biological activity of FIEF. To test oup hypothesis, w? designed a cell poliferatim assay in lhich early passage rat lmg fitilasts (RLFs) wsregtum in 10% FBS fcr24h,thenwashedwith serui+freemdiunbefcre tiing a-free defined lrediun (ON) containing insulin and transferrin. The grokh mrdiun was supplenanted with varying cmcentratims of either hutIvl Ax;F, humn a-M cr a cmhinatim. After 3days,tk cellswere harvested by trypsinizatim and cells ware emnwrated using a Cculter Ccunter, A cmcmtratim&pendmt gmth curvewas deomstrated in OMfcr KlGFalcne,with near fmxinel stiiwlatim reached at 15-20 w/ml KGF. o-M alme had no effect m cell croliferatim. tla*wff. a-M mhanced FDGF-stirmlatei proliferatim 6C-&X in the cresenceof15ng/ml Rx;F,with aaoximalenhancemntcccurt-inghaken D3-lCOw/mla+i. In addition, fractions ofrataaw itimedimiiun containingcatplexes enhanced fitxublast~liferatim by-40% above cmtrol tiiun. F~e,we&nmstrated thatRlFpas.sess hi@affinityrecepks fcr hurm FDGFaswellas fcr the ~ease-bound fumof iuran *2-!4. We postulate that a-M mhances FDGF-stimtlated gruvth by regulating the release ofRx;F ftunC complexes, u-via theaM cell-surface receptors m the fitrcblasts.
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MODULATION OF NK ACTIVITY BY MONOCYTES: EFFECTS OF MONOCYTE (MO) ADHERENCE, PGE AND INTERLEUKIN-1. E.T. Bloom and J.T. Babbitt. VA Med Ct?'West LA, Los Angeles, CA 90073, and UCLA SThMed, Los Angeles, CA 90024. We have reported that MO both up- and down-regulate human NK cell activity and that IL-l and IL-2 are involved in the augmentation. We have now evaluated the roles of MO adherence, exogenous IL-1 and exogenous PGE in the regulation of human NK activity by MO. MO isolated b$ Percoll centrifugation did not modulate NK activity while MO isolated by adherence usually exhibited increased augmentation (eg. from O-100% increase) with increased period of adherence (up to 1 hr). Rarely, however, increased suppression (eg. from 50%-O% increase) was observed with increased time of adherence. Increased augmentation correlated with increased IL-1 and decreased PGE production. We then examined (1) the roles of exogenous IL-1 and PGE2 in the absence or presence of MO in the NK assay z$stem; (2) the participation of PGE by pretreating MO with 10 M indomethacin: and (31 the oa F.'ticioation of MO membrane bound IL-1 by fixing MO .with i% paraformaldehyde. Exogenous PGE (0.6-3 no/ml1 SUDDreSSed NK activitv. while exoaenous IL-l-g (100-5OO&u/~lj had no detectable effect. Howeve;, NK-MO cell mixture experiments revealed that PGE2 is involved in the suppression of human NK activity by MO, and that a functional balance between IL-l and PGE, determines whether up- or downregulation is observed. The=data are consistent with the interoretation that membrane-associated IL-1 is the imoortant IL-1 moiety for the augmentation of human NK activity by MO.
PERILYNPHATIC INJECTION OF IL-4 WITH OR WITHOUT OTHER LYMPHOKINES ACTIVITIES REJECTION OF POORLY IMMUNOGENIC MOUSE TLlM0RS.M.C. Bosco. M. Giovarelli. and G. Forni. Department of Microbiology, Universitv of Turin, Turin, Italv. IL-4 has-bee" show" tb activate.B lymphocytes, MO and CTL It was therefore decided to study the ability of perilymphatically injected IL-4 to inhibit the growth of murine tumors. Ten daily injections of 0.1-100 pg rIL-4 (Immunex) induced dose dependent inhibition of the growth of a poorly immunogenic sarcoma (GE-2) raised by methylcolanthrene in Balb/c mice. Association of rIL-la, a synthetic "onpeptide (Sclavo), or rIL-2 created a helper system capable of recruiting a" effective antitumor reactivity. This reaction is mediated by granulocytes, MO, NK cells, and T-lymphocytes: the tumor area is infiltrated by mononuclear cells and granulocytes (mostly eosinophils), which form close contacts between each other and with the neoplastic cells. The draining lymph nodes display marked expansion of the cortical follicles and massive infiltration into the medulla of histiocytes, foreign-body giant cells, plasma cells and immunoblasts, joined together by lymphocytes and by gra"ulocytes. The local activity becomes systemic and confers a specific immune memory. The growth of a second, contralateral inoculum was inhibited in a significant number of animals. A more comprehensive assessment of the extent to this reaction was made by studying its inhibition of the growth of a spontaneous non-immunogenic and metastasizing mouse breast tumor (TS/A).
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ON CYTOKINES
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INFECTION WITH MYCOBACTERIUM AVIUM COMPLEX (MAC) STIMULATES FIBROBLASTS (FI) TO PRODUCE GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR AND INTERFERON-BETA. L. Bermudez. Kuzell Institute, San Francisco, CA 94115. MAC is the most common bacterial agent isolated from AIDS patients. In AIDS, MAC is associated with disseminated disease, and is found invading and replicating within macrophages and monocytes. Early in the infection, MAC could invade Fi and endothelial cells. Murine (L929) Fi cell line was infected with a serotype 1 of MAC. The duplication time intracellularly was 40 hours. Progressive intracellular growth ultimately resulted in the destruction of infected cells. Supernatant obtained from infected Fi contained IFN)3 and GM-CSF [50+12 ng and 26090 pg. respectively by 18 hours). IFN)3 could be detected by 3 hours after infection, while GM-CSF was first detected by 6 hours. Treatment of infected Fi with TNF, (10 to 103 U/ml) and GM-CSF (10 to 102 U/ml) was associated with inhibition of the intracellular growth of MAC (62+7% and 60+4%. respectively). Release of cytokines from fibroblasts infected with MAC can subsequently activate macrophages and NK cells, initiating the host defense against the infection.
ENHANCED IN VITRO RECRUITERNT OF LAK ACTIVITY IN IN VIVO PRIMED LWOCYTES. R.L.H. Bolhuis, G. Stoter and C.Lamers. Daniel Den Hoed Cancer Center, Rotterdam, lhe Netherlands. NK and LAK activities of lymfocytes from patients receiving adoptive cellular immunotherapy: in vivo IL-2 activated lymfocytes show enhanced recruitement 3 ljXc activity upon in vitro 11-Z activation, but no dependency on 11-2. Cryopreservation only slightly lowers NK- and LAK activities of in vitro 11-2 activated MNC obtained from in vivo 11-2 primed cancer patients. NK and LAK activities were decreased following cryopreservation but activity was reconstituted by resting the MNC for eighteen hours. Presence of IL-2 during the cytolitic assay increased in vivo induced but not in vitro induced LAK activity. Kinetic analysis of the 11-Z enhanced cytolysis demonstrated that the enhancement of lytic activities recycled from recruitement of LAK activity but not from an intrinsic dependency of lymfocytes on 11-2. Conclusions: l-Addition of 11-2 during cytolysis for imnuneparameter monitoring may lead to an overestimate of lytic activity. e-The data suggest that prolonged in vivo administration of low-dose 11-2 will maintain maximum levels of in vivo LAK activity in the patient.
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MACROPHAGE-COLONY STIMULATING FACTOR (M-CSF) PRIMES HUMAN MACROPHAGES (MO) FOR ACTIVATION BY OTHER CYTOKINES. L. Bermudez. Kuzell Institute, San Francisco. CA 94115. MO stimulation is a chief step in the host defense against intracellular infectious microorganisms. We examined the sequential stimulation of human monocytederived MO using Mycobacterium auium (MAC) serotype 1 as a model of intracellular Infection. MO cultured in vitro for 7 days were infected with MAC and treated with recombinant IFNy, M-CSF, GM-CSF. TNF and IL-4, using different doses and sequences of stimulation. After four days, monolayers were lysed and the number of viable intracellular bacilli were quantitated after plating. M-CSF showed to be an excellent primer for further activation with lower concentrations of GM-CSF and TNF [85-o% and 88?4% of intracellular killing of MAC, respectively, compared to 26+4% and 29*7% of each alone at the same concentration (1 U/ml)). When used in inverse sequencing (TNF or GM-CSF, followed by M-CSF), the resultant intracellular killing was significantly smaller (1726% for GM-CSF/M-CSF and 3ti4% for TNF/M-CSF). These results suggest that combined treatment of human MO with cytokines may reproduce the physiologic sequence of cell activation.
AL~ROGU)BIIlINSBICWYCE~-~FUAW~LIFERRTIONOF~ FIWBLAm IN VITlXl. J.C. Banner, hi. Hoffnan, A. B&e& A. Osunic-Vat-gas tiA.R.Brod.Lab.o'fPul Pthob'l Nat1 1st fEni Hlth mg. PK., NC. ZG. ofk&l., ihi:. ofoK, Gi Hill; NC. 3??7K-? Alpha-nacrcglcbulins (a-k) secreted byalveolartnacrophages areplateletderived q&h factcr (KHZ) binding pnteins that cq&e with cell-surface recepks cn rat lung fib&lasts fcr KGF (ker. J. Resp. Cell bl. Biol., In Press, 1959). Since au-e than 50% of the FDGF secreted by these lug nocro&qesiscarplexeitoa+i,amiPDGFisaccnpetmcefactati fikcblast gru&h, we p3stulatezi that a-& mxlulate the biological activity of FIEF. To test oup hypothesis, w? designed a cell poliferatim assay in lhich early passage rat lmg fitilasts (RLFs) wsregtum in 10% FBS fcr24h,thenwashedwith serui+freemdiunbefcre tiing a-free defined lrediun (ON) containing insulin and transferrin. The grokh mrdiun was supplenanted with varying cmcentratims of either hutIvl Ax;F, humn a-M cr a cmhinatim. After 3days,tk cellswere harvested by trypsinizatim and cells ware emnwrated using a Cculter Ccunter, A cmcmtratim&pendmt gmth curvewas deomstrated in OMfcr KlGFalcne,with near fmxinel stiiwlatim reached at 15-20 w/ml KGF. o-M alme had no effect m cell croliferatim. tla*wff. a-M mhanced FDGF-stirmlatei proliferatim 6C-&X in the cresenceof15ng/ml Rx;F,with aaoximalenhancemntcccurt-inghaken D3-lCOw/mla+i. In addition, fractions ofrataaw itimedimiiun containingcatplexes enhanced fitxublast~liferatim by-40% above cmtrol tiiun. F~e,we&nmstrated thatRlFpas.sess hi@affinityrecepks fcr hurm FDGFaswellas fcr the ~ease-bound fumof iuran *2-!4. We postulate that a-M mhances FDGF-stimtlated gruvth by regulating the release ofRx;F ftunC complexes, u-via theaM cell-surface receptors m the fitrcblasts.
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MODULATION OF NK ACTIVITY BY MONOCYTES: EFFECTS OF MONOCYTE (MO) ADHERENCE, PGE AND INTERLEUKIN-1. E.T. Bloom and J.T. Babbitt. VA Med Ct?'West LA, Los Angeles, CA 90073, and UCLA SThMed, Los Angeles, CA 90024. We have reported that MO both up- and down-regulate human NK cell activity and that IL-l and IL-2 are involved in the augmentation. We have now evaluated the roles of MO adherence, exogenous IL-1 and exogenous PGE in the regulation of human NK activity by MO. MO isolated b$ Percoll centrifugation did not modulate NK activity while MO isolated by adherence usually exhibited increased augmentation (eg. from O-100% increase) with increased period of adherence (up to 1 hr). Rarely, however, increased suppression (eg. from 50%-O% increase) was observed with increased time of adherence. Increased augmentation correlated with increased IL-1 and decreased PGE production. We then examined (1) the roles of exogenous IL-1 and PGE2 in the absence or presence of MO in the NK assay z$stem; (2) the participation of PGE by pretreating MO with 10 M indomethacin: and (31 the oa F.'ticioation of MO membrane bound IL-1 by fixing MO .with i% paraformaldehyde. Exogenous PGE (0.6-3 no/ml1 SUDDreSSed NK activitv. while exoaenous IL-l-g (100-5OO&u/~lj had no detectable effect. Howeve;, NK-MO cell mixture experiments revealed that PGE2 is involved in the suppression of human NK activity by MO, and that a functional balance between IL-l and PGE, determines whether up- or downregulation is observed. The=data are consistent with the interoretation that membrane-associated IL-1 is the imoortant IL-1 moiety for the augmentation of human NK activity by MO.
PERILYNPHATIC INJECTION OF IL-4 WITH OR WITHOUT OTHER LYMPHOKINES ACTIVITIES REJECTION OF POORLY IMMUNOGENIC MOUSE TLlM0RS.M.C. Bosco. M. Giovarelli. and G. Forni. Department of Microbiology, Universitv of Turin, Turin, Italv. IL-4 has-bee" show" tb activate.B lymphocytes, MO and CTL It was therefore decided to study the ability of perilymphatically injected IL-4 to inhibit the growth of murine tumors. Ten daily injections of 0.1-100 pg rIL-4 (Immunex) induced dose dependent inhibition of the growth of a poorly immunogenic sarcoma (GE-2) raised by methylcolanthrene in Balb/c mice. Association of rIL-la, a synthetic "onpeptide (Sclavo), or rIL-2 created a helper system capable of recruiting a" effective antitumor reactivity. This reaction is mediated by granulocytes, MO, NK cells, and T-lymphocytes: the tumor area is infiltrated by mononuclear cells and granulocytes (mostly eosinophils), which form close contacts between each other and with the neoplastic cells. The draining lymph nodes display marked expansion of the cortical follicles and massive infiltration into the medulla of histiocytes, foreign-body giant cells, plasma cells and immunoblasts, joined together by lymphocytes and by gra"ulocytes. The local activity becomes systemic and confers a specific immune memory. The growth of a second, contralateral inoculum was inhibited in a significant number of animals. A more comprehensive assessment of the extent to this reaction was made by studying its inhibition of the growth of a spontaneous non-immunogenic and metastasizing mouse breast tumor (TS/A).