- Email: [email protected]
Cytokine regulation of human immunodeficiency virus expression in monocytic cells
THIRD
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
/ 513
379
382
CYTOKINES REGULATION OF INTERLEUKIN-6 (IL-6) AND SOLUBLE IL-6 RECEPTOR (.S.IL-6R) SECRETION BY HUMAN MESANGIAL CELLS. A.Moutabarrik. M.Ishibashi. H. Kameoka, Y.Kokado, S.Takahara,.Y.Takano T.Sonoda and A.Okuyama. Dept Of Urology. Osaka UAiversity. IL-6 affects growth and differentiation of a wide spectrum of cells. IL-6 receptor is expressed on many of those cells. S.IL-CRis secreted by many cells expressing IL-6R. Human mesangial cells HMC are an important source of IL-6 and express IL-6R. The influence of lipopolysaccarides(LPP.1, recombiant human IL-18 (rh IL-lB), TNF (rh TNFa), and IFN (rh IFN’I) on the production of IL-6 and S.IL-6 R was investigated. The addition of O.l-lOOpg/ml of LPS, O.l-lOOu/ml of rhIL-1!3, or O.l-lOOng/ml of rhTNFa to culture of HMC monolaver caused a doserelated increase in IL-6 secretion as detected by ELISA and bioassay using MH60 cell line, after 4, 10, 20, and 30 hours culture. In co trast to LPS, rh IL-18, and rhTNFa, the use of 10 !I u/ml of rhIFNY caused no increase in IL-6 secretion and modestlv potentiates the effect of LPS, rhIL-1!3, and rhT@o In the same manner, unstimulated HMC secrete a small amount of S.IL-6R in the culture supernatant, LPS, rhIL-1!3, and rhTNFa treatment of HMC for 6H increase significantly the release of S.IL-6R as detected by an ELISA assay, (respectively 373 +23, 520 *43, 253 *75 pg/ml vs 196t 80 of the control pc 0.05). IL-6 and S.IL-6R are may be mediators involved in glomerular inflammatory processes.
INVlVOROLESOFIL-laANDIL-1B. Ivan G. Otternesr, Marcia L. Bliven. Isabel Carreras* and Jean D. Sipe.* Central Research Division, Pfizer Inc. Groton, CT 06340and *Dept Biochemistry, Boston University Medical School, Boston, MA 02118. The relative importance of the two cytokiner IL-la and IL-l p in viva has been debated. We have prepared blocking antibodiesagainst murine IL-la and IL-lfland validated them in viva by determining theamountsof antibody that will block IL-6 production after subcutaneous (s.c.) administration of 300 ng IL-la or IL-l& Anti-IL-laantibodyinhibited IL-la, but not IL-lo; the After injection of turpentine, conversespecificitywas found for anti-IL-lb. weight loss, IL-6 production, SAP production have been shown to be inhibited by the IL-1 receptor antagonist IL-1 RA (Gerrhenwald eta/, PNAS 87,496, 1990). Using the same murine system, we administered intraperitoneally normal sera or blocking amounts of anti-IL-la. anti-IL-ll3 or anti-IL-la + antiIL-1 B antisera. 24 hr later the mice were weighed, and turpentine given S.C. An additional 24 hrs later, the mice were weighed again, bled, and plasma SAA and IL-6 determined. SAA and IL-6 levels in plasma were inhibited by anti-IL-la + anti-IL-lpantibody, but not byanti-IL-laor IL-lpseparately. Weight losswascompletely reversed by anti-IL-1Bantibodyalone; therewas no effect of anti-IL-la alone nor any additional effect with the combination of uc p antibodies. Although both IL-la and IL-lb can cause weight loss and fever, our results (here and Long et al, Am JPhysiol259, R724, 1990) indicate that IL-lb is the principle form of IL-1 in these two responses. By contrast, both IL-laand IL-IBare present in ampleamountstoindependentlyelicit the SAA and IL-6 response.
380
383
DIETARY SUPPLEMENTATION WITH GAMMA-LINOLENIC AND PARAMETERS OF CELL-MEDIATED IMMUNITY.
ACID
(GLA)
mediated immune parameters. This effect is only partially influenced by prostaglandin metabolism. The type of fatty acid in the diet may modulate an individual’s response to a given stimulus.
IRON CARRIER PROTEINS (ICP): EVIDENCE FOR IMPORTANT IMMUNOMOOULATORY ROLES IN VIVO Walter Pierpaoli, Institute for Biomedical Research, Quartino, Switzerland; Paolo Neri and Annalisa Santucci, Dipartimento di Biologia Molecolare, Universita di Siena, Italy. In the last decade we have proposed the concept and provided the experimental evidence that the problems ensuing after tran splantation of histoincompatible bone marrow depend mainly not on immunogenetic diversity between donor and recipient of bone marrow but rather on a deficient marrow engraftment. It is thus possible to obtain permanent allogeneic hemopoietic chimerism in mice provided we supply the immunosuppressed recipient with factors endogenous to the bone marrow, which greatly facilitate the engraftment of H-2 incompatible marrow. A very long procedure of progressive fractionation of marrow components has now allowed to identify a glycoprotein, namely transferrin, which, when given in the course of transplantation, induces a rapid engraftment of allogeneic bone marrow and permanent chimerism, without any symptoms of "graft-versus-host disease". It seems thus that ICP are able, with a mechanism still to be clarified, to operate changes in the host or in the donated marrow containing the whole repertoire of cytotoxic T cells, which result into successful allogeneic engraftment. These results lend support to our early suggestion that the problem of bone marrow transplantation is more hematologic than inmmnologic in its character and that intervention with endogenous bone marrow agents, now identified as transferrins, can help to solve this basic biological and medical problem.
381
384
DIETARY SUPPLEMENTATION WlTH GAMMA-LINOLENIC ACID (GLA) AND SYNTHESIS OF IL-1 AND TNF BY PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC). J. L. Nerad. S. N. Mevdani, and C. A.
CYTOKINE REGULATION OF HUMAN IMMUNODEFICIENCY VIRUS EXPRESSION IN MONOCYTIC CELLS. a Poli. A f Kinter. S J Juste em. P. Bressler. P. Biswas and A.S. Fu i. NIAID, NIH, BethEda, MD 20892. Cytokines such as tumor necrosis factor (TNF) induce the expression of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), in both infected T and monocytic cells via activation of the cellular transcription factor NF-kB. Other cytokines such as interleukin-6 (IL-6) are also upregulators of virus production, but their effect is restricted to the monocytic lineage and is predominantly exerted at a post-transcriptional level. More recently we found that transforming growth factor beta (TGF-P) potently suppressed HIV expression at the transcriptional level in the chronically HIV infected Ul cell line (derived from the U937 promonocytic cell line) stimulated with PMA. In addition, TGF-P suppressed virus production in primary monocyte-derived macrophages (MDM) previously infected with HIV. However, stimulation of both U937 cells or MDM with TGF-p before HIV infection resulted in increased virus production. Because the production of several of these cytokines is elevated in HIV infected individuals, it is likely that they may play an important role as modulators of virus expression in vivo.
1. L.
Nerad. S. N. Mevdani. and C. A. Dinarello, New England Medical Center; USDA Human Nutrition Research Center on Aging. Tufts University, Boston, MA, USA, 02111. GLA has been implicated as having therapeutic benefit in chronic inflammatory disorders. IL-2, mitogen-stimulated lymphocyte proliferation, and delayed-type hypersensitivity skin reactions (DTH) are indicators of cellmediated immune function. We examined whether dietary supplementation with GLA influenced in vitro and in viva cell-mediated immunity. In a double blind trial, 8 healthy volunteers added 1925 trig of GLA and 4 added 5-9 gm olive oil/coconut oil (00 capsules each day for 12 weeks to their regular diets. Peripheral blood mononuclear cells were isolated and stimulated with phytohemagglutinin. Cytokines were measured by ELISA or RIA, lymphocyte proliferation was measured by tritiated-thymidine uptake, and DTH evaluated by Multi-Test-CM1 (Connaught-Merieux). After 12 weeks of supplementation with GLA, there was decreased production of PHA stimulated IL-l& PGE2, and lymphocyte proliferation. In both the OC and GLA groups, PHA-stimulated IL-2 production was increased after 12 weeks. No change in PGEl production was noted in either group. DTH results are reported as number of positive antigens (antigen score) and total diameter of induration (cumulative score, mm). After 12 weeks of supplementation, there was no difference between groups in antigen score. However, an increase in the cumulative rare hean+xm) in the GLA group (6.3~1.6 mm to 22.1i6.4 mm) and a decrease in the OC group f25.2k5.8 mm to 15.Ok4.4 nun) were noted. We conclude that dietary supplementation with specific oils influence cell-
Dinarello. New England Medical Center; USDA Human Nutrition Research Center on Aging, T&s University, Ftoston,MA, USA, 02111. GLA has been implicated as having therapeutic benefit in inflammatory disorders. IL-1 and TNF are two cytokines shown to be associated with inflammatory processes. We examined whether dietary supplementation with GLA influenced in vitro cytokine production. In a double blind trial, 8 healthy volunteers added 1925 rng of GLA and 4 added 5-9 pm olive oil/co&nut oil (CC) capsules each day for 12 weeks to their regular diets. PBMC were isolated and stimulated with endotoxin. Staohvlococcus. IL-1 0. or IL-2 with or without indomethacin. IL-1 6 stimuiated’IL~1 alpha production increased from 2238+/264 pg/ml @aseline) to 4224+/-617 pg/ml in the GLA group after 8 weeks of supplementation and from 1068+/-182 pg/ml to 4570+/-1205 pg/ml in the OC group after 12 weeks of supplementation. Similar results were seen with TNF production stimulated by IL-IL? and endotoxin, and IL-l alpha and TNF production after stimulation with IL-2. Twenty weeks after the end of supplementation, cytokine production had returned to the presupplement level. The increased production of IL-1 and ‘INF were accompanied by an increase in spontaneous PGE2 production only in the GLA-treated group and indomethacin only partially reduced the increase in cytokine production observed in the two groups. We conclude that dietary supplementation with GLA and olive oil/coconut oil can enhance cytnkine-induced cytokine production. This effect is only partially influenced by prostaglandin metabolism. The type of stimulus elliciting the inflammatory process may determine the effectiveness of putative anti-inflammatory agents such as GLA.
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
/ 513
379
382
CYTOKINES REGULATION OF INTERLEUKIN-6 (IL-6) AND SOLUBLE IL-6 RECEPTOR (.S.IL-6R) SECRETION BY HUMAN MESANGIAL CELLS. A.Moutabarrik. M.Ishibashi. H. Kameoka, Y.Kokado, S.Takahara,.Y.Takano T.Sonoda and A.Okuyama. Dept Of Urology. Osaka UAiversity. IL-6 affects growth and differentiation of a wide spectrum of cells. IL-6 receptor is expressed on many of those cells. S.IL-CRis secreted by many cells expressing IL-6R. Human mesangial cells HMC are an important source of IL-6 and express IL-6R. The influence of lipopolysaccarides(LPP.1, recombiant human IL-18 (rh IL-lB), TNF (rh TNFa), and IFN (rh IFN’I) on the production of IL-6 and S.IL-6 R was investigated. The addition of O.l-lOOpg/ml of LPS, O.l-lOOu/ml of rhIL-1!3, or O.l-lOOng/ml of rhTNFa to culture of HMC monolaver caused a doserelated increase in IL-6 secretion as detected by ELISA and bioassay using MH60 cell line, after 4, 10, 20, and 30 hours culture. In co trast to LPS, rh IL-18, and rhTNFa, the use of 10 !I u/ml of rhIFNY caused no increase in IL-6 secretion and modestlv potentiates the effect of LPS, rhIL-1!3, and rhT@o In the same manner, unstimulated HMC secrete a small amount of S.IL-6R in the culture supernatant, LPS, rhIL-1!3, and rhTNFa treatment of HMC for 6H increase significantly the release of S.IL-6R as detected by an ELISA assay, (respectively 373 +23, 520 *43, 253 *75 pg/ml vs 196t 80 of the control pc 0.05). IL-6 and S.IL-6R are may be mediators involved in glomerular inflammatory processes.
INVlVOROLESOFIL-laANDIL-1B. Ivan G. Otternesr, Marcia L. Bliven. Isabel Carreras* and Jean D. Sipe.* Central Research Division, Pfizer Inc. Groton, CT 06340and *Dept Biochemistry, Boston University Medical School, Boston, MA 02118. The relative importance of the two cytokiner IL-la and IL-l p in viva has been debated. We have prepared blocking antibodiesagainst murine IL-la and IL-lfland validated them in viva by determining theamountsof antibody that will block IL-6 production after subcutaneous (s.c.) administration of 300 ng IL-la or IL-l& Anti-IL-laantibodyinhibited IL-la, but not IL-lo; the After injection of turpentine, conversespecificitywas found for anti-IL-lb. weight loss, IL-6 production, SAP production have been shown to be inhibited by the IL-1 receptor antagonist IL-1 RA (Gerrhenwald eta/, PNAS 87,496, 1990). Using the same murine system, we administered intraperitoneally normal sera or blocking amounts of anti-IL-la. anti-IL-ll3 or anti-IL-la + antiIL-1 B antisera. 24 hr later the mice were weighed, and turpentine given S.C. An additional 24 hrs later, the mice were weighed again, bled, and plasma SAA and IL-6 determined. SAA and IL-6 levels in plasma were inhibited by anti-IL-la + anti-IL-lpantibody, but not byanti-IL-laor IL-lpseparately. Weight losswascompletely reversed by anti-IL-1Bantibodyalone; therewas no effect of anti-IL-la alone nor any additional effect with the combination of uc p antibodies. Although both IL-la and IL-lb can cause weight loss and fever, our results (here and Long et al, Am JPhysiol259, R724, 1990) indicate that IL-lb is the principle form of IL-1 in these two responses. By contrast, both IL-laand IL-IBare present in ampleamountstoindependentlyelicit the SAA and IL-6 response.
380
383
DIETARY SUPPLEMENTATION WITH GAMMA-LINOLENIC AND PARAMETERS OF CELL-MEDIATED IMMUNITY.
ACID
(GLA)
mediated immune parameters. This effect is only partially influenced by prostaglandin metabolism. The type of fatty acid in the diet may modulate an individual’s response to a given stimulus.
IRON CARRIER PROTEINS (ICP): EVIDENCE FOR IMPORTANT IMMUNOMOOULATORY ROLES IN VIVO Walter Pierpaoli, Institute for Biomedical Research, Quartino, Switzerland; Paolo Neri and Annalisa Santucci, Dipartimento di Biologia Molecolare, Universita di Siena, Italy. In the last decade we have proposed the concept and provided the experimental evidence that the problems ensuing after tran splantation of histoincompatible bone marrow depend mainly not on immunogenetic diversity between donor and recipient of bone marrow but rather on a deficient marrow engraftment. It is thus possible to obtain permanent allogeneic hemopoietic chimerism in mice provided we supply the immunosuppressed recipient with factors endogenous to the bone marrow, which greatly facilitate the engraftment of H-2 incompatible marrow. A very long procedure of progressive fractionation of marrow components has now allowed to identify a glycoprotein, namely transferrin, which, when given in the course of transplantation, induces a rapid engraftment of allogeneic bone marrow and permanent chimerism, without any symptoms of "graft-versus-host disease". It seems thus that ICP are able, with a mechanism still to be clarified, to operate changes in the host or in the donated marrow containing the whole repertoire of cytotoxic T cells, which result into successful allogeneic engraftment. These results lend support to our early suggestion that the problem of bone marrow transplantation is more hematologic than inmmnologic in its character and that intervention with endogenous bone marrow agents, now identified as transferrins, can help to solve this basic biological and medical problem.
381
384
DIETARY SUPPLEMENTATION WlTH GAMMA-LINOLENIC ACID (GLA) AND SYNTHESIS OF IL-1 AND TNF BY PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC). J. L. Nerad. S. N. Mevdani, and C. A.
CYTOKINE REGULATION OF HUMAN IMMUNODEFICIENCY VIRUS EXPRESSION IN MONOCYTIC CELLS. a Poli. A f Kinter. S J Juste em. P. Bressler. P. Biswas and A.S. Fu i. NIAID, NIH, BethEda, MD 20892. Cytokines such as tumor necrosis factor (TNF) induce the expression of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), in both infected T and monocytic cells via activation of the cellular transcription factor NF-kB. Other cytokines such as interleukin-6 (IL-6) are also upregulators of virus production, but their effect is restricted to the monocytic lineage and is predominantly exerted at a post-transcriptional level. More recently we found that transforming growth factor beta (TGF-P) potently suppressed HIV expression at the transcriptional level in the chronically HIV infected Ul cell line (derived from the U937 promonocytic cell line) stimulated with PMA. In addition, TGF-P suppressed virus production in primary monocyte-derived macrophages (MDM) previously infected with HIV. However, stimulation of both U937 cells or MDM with TGF-p before HIV infection resulted in increased virus production. Because the production of several of these cytokines is elevated in HIV infected individuals, it is likely that they may play an important role as modulators of virus expression in vivo.
1. L.
Nerad. S. N. Mevdani. and C. A. Dinarello, New England Medical Center; USDA Human Nutrition Research Center on Aging. Tufts University, Boston, MA, USA, 02111. GLA has been implicated as having therapeutic benefit in chronic inflammatory disorders. IL-2, mitogen-stimulated lymphocyte proliferation, and delayed-type hypersensitivity skin reactions (DTH) are indicators of cellmediated immune function. We examined whether dietary supplementation with GLA influenced in vitro and in viva cell-mediated immunity. In a double blind trial, 8 healthy volunteers added 1925 trig of GLA and 4 added 5-9 gm olive oil/coconut oil (00 capsules each day for 12 weeks to their regular diets. Peripheral blood mononuclear cells were isolated and stimulated with phytohemagglutinin. Cytokines were measured by ELISA or RIA, lymphocyte proliferation was measured by tritiated-thymidine uptake, and DTH evaluated by Multi-Test-CM1 (Connaught-Merieux). After 12 weeks of supplementation with GLA, there was decreased production of PHA stimulated IL-l& PGE2, and lymphocyte proliferation. In both the OC and GLA groups, PHA-stimulated IL-2 production was increased after 12 weeks. No change in PGEl production was noted in either group. DTH results are reported as number of positive antigens (antigen score) and total diameter of induration (cumulative score, mm). After 12 weeks of supplementation, there was no difference between groups in antigen score. However, an increase in the cumulative rare hean+xm) in the GLA group (6.3~1.6 mm to 22.1i6.4 mm) and a decrease in the OC group f25.2k5.8 mm to 15.Ok4.4 nun) were noted. We conclude that dietary supplementation with specific oils influence cell-
Dinarello. New England Medical Center; USDA Human Nutrition Research Center on Aging, T&s University, Ftoston,MA, USA, 02111. GLA has been implicated as having therapeutic benefit in inflammatory disorders. IL-1 and TNF are two cytokines shown to be associated with inflammatory processes. We examined whether dietary supplementation with GLA influenced in vitro cytokine production. In a double blind trial, 8 healthy volunteers added 1925 rng of GLA and 4 added 5-9 pm olive oil/co&nut oil (CC) capsules each day for 12 weeks to their regular diets. PBMC were isolated and stimulated with endotoxin. Staohvlococcus. IL-1 0. or IL-2 with or without indomethacin. IL-1 6 stimuiated’IL~1 alpha production increased from 2238+/264 pg/ml @aseline) to 4224+/-617 pg/ml in the GLA group after 8 weeks of supplementation and from 1068+/-182 pg/ml to 4570+/-1205 pg/ml in the OC group after 12 weeks of supplementation. Similar results were seen with TNF production stimulated by IL-IL? and endotoxin, and IL-l alpha and TNF production after stimulation with IL-2. Twenty weeks after the end of supplementation, cytokine production had returned to the presupplement level. The increased production of IL-1 and ‘INF were accompanied by an increase in spontaneous PGE2 production only in the GLA-treated group and indomethacin only partially reduced the increase in cytokine production observed in the two groups. We conclude that dietary supplementation with GLA and olive oil/coconut oil can enhance cytnkine-induced cytokine production. This effect is only partially influenced by prostaglandin metabolism. The type of stimulus elliciting the inflammatory process may determine the effectiveness of putative anti-inflammatory agents such as GLA.