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Human vascular smooth muscle cells produce intracellular IL1 receptor antagonist, IL1β precursor, and IL1α
CYTOKINE,
542 I Abstracts
Vol. 6: No. 5 (September
1994: 539-582)
A19 ACTIVATION OF PERIPHERAL BLOOD T CELLS THROUGH LIGANDS OF THE TNF FAMILY. S. Wiley, C. Smith, M. Alderson, and R. Goodwin. lmmunex Corporation, 51 University SL, Seattle WA 98101. Our group is testing the potential of TNF ligand family members lo transduce signals to their host cells independent of their cognate receptors. The TNF ligand family includes several related proteins which can be found on the surface of peripheral blood T (PBT) cells subsequent to stimulation with antiCD3. These proteins include CD30 ligand (CD30L), CD40 ligand (CD40L). Fas ligand, and TNFa. The surface forms of these molecules are type II Vansmembrane proteins with their aminotermini inside of the cell. The amino acid sequences of individual TNF ligand family members' cytoplasmic domains are conserved between species, but are divergent amongst different family members. The percent amino acid conservation of the cytoplasmic domains between the mouse and human forms of CD40L, TNFa dnd CD30L are 82%, 86%, and 61%, respectively. This inter-species sequence conservation strongly suggests some functional role for the cyloplasmic domains of the ligands. We have shown that a monoclonal antibody against human CD30L is able to stimulate PBTs in the presence of anti-CD3. This phenomenon is not a property of CD30L alone, since stimulation using these conditions has been produced with monoclonal antibodies against the CD40L and with a fusion protein consisting of the Fc domain and the extracellular domain of the LTP receptor. These results strongly suggest that some of the TNF family ligands themselves can transduce stimulatory signals lo their host cells, thereby blurring the distinction between receptor and ligand. Further experiments are underway to measure cytokine production and regulation of cell surface markers in PBT cells stimulated with anti-CDBOL and anti-CDS, and lo search for additional biological effects mediated by other TNF ligand family members.
P2 October 4: Lymphokinedcytokines
in Hematopoiesis
A20
A22 Stimulation of Megakaryocytopoiesis Thrombopoiesis by the c-Mpl Frederic
J.
deSauvage
& Dan L.
and ligand
Eaton
The Departments of Molecular Biology & Cardiovascular Research, Genentech Inc., S. San Francisco, CA 94080 Physiological platelet synthesis is thought to require humoral activities known as meg-CSF and thrombopoietin which promote proliferation and maturation of megakaryocytic cells, respectively. Here, we describe the purification and molecular cloning of a meg-CSF/TPO-like protein that is present in plasma of irradiated pigs. This orotein binds to. and activates. c-mol. a member of thk cytokine receptor superfamily: The isolated mpl ligand is a novel cytokine with a unique structure; the amino terminal domain is homologous to erythropoietin and the C-terminal glycosylated domain is unrelated to any known protein. In vitro and in vivo experiments with recombinant mpl ligand indicate that it has both megakaryocyte colony-stimulating activity and thrombopoietin activity, and therefore corresponds to the long sought platelet growth factor, thrombopoietin (TPO).
Effective Cytokine Gene Transfer to Human Bone Marrow Stromal Cells using El Deleted Adenoviral Vectors. R. Foley, T. Braciak, C. Addison, C. Brazolot-Millan, I. Walker, R. Carter, F. Graham, J. Gauldie, McMaster University, Hamilton, Ontario, Canada L8N 325 An adenoviral cytokine gene transfer system was used in human long term bone marrow culture to demonstrate the feasibility of genetically manipulating adherent (stromal) cells to produce functional cytokine at high levels over a period of days. Human bone marrow
samples from healthy
marrow
transplant
donors
were maintained
in
modified Dexter’s long term culture for the generation of an adherent layer representative of the marrow stroma. Adenoviral constructs designed to transfer the gene for P-galactosidase and murine interleukin-6 (IL-4) were used to infect adherent layers. Adenoviral vectors were constructed with cDNA inserted into the El region of the viral genome under the transcriptional control of a human
cytomegalovirus promoter (hCMV). Our results demonstrate effective gene transfer and expression of functional protein at high levels over a period
the range
of at least 10 days in vitro.
of micrograms
response infection)
relationship and cytokine
cytokine
gene transfer
Levels of functional
per millilitre
of culture
IL-6 were in
media.
A dose
between viral concentration (multiplicity of production was seen. We consider adenoviral
in adherent
human marrow
culture
useful in evaluating these relationships and the modulation maturation. (Supported by MRC and NC1 Canada)
potentially of stem cell
A23
A21 EFFECT OF ANTI-CD3 INJECTION ON HEMATOPOIESIS AND IL-3. INDUCED HISTAMWE AND IL-6 PRODUCTION BY PROGENITORS. M. Dy, V. Salaiin , A. Ben Amor, L. Chatenond, C. Le Bousse, E. Schneider. CNRS URA 1461- INSERM U25, Hapita Necker, Paris, INSERM U 268 , Villejnif, FRANCE. Hematopoietic progenitors respond to IL-3 in vitro by producing large nmounts of histamine and IL-6. This has also be demonstrated in viva during antigenic challenge of immunized mice where IL-3 can be detected in the circulation. Herein, we provide evidence for a similar effect of anti-CD3 injection into normal mice, i.e. the appearance of circulating IL-3 accompanied by enhanced activity of histidine decarboxylase. the histamine-forming enzyme, in bone marrow and spleen. In addition, we show that the frequency of CFC and BFU-E in the bone marrow increases substantially during the first days following anti-CD3 administration, while the effect of IL-3 on the ex viva production of histamine and IL-6 by bone marrow cells is also enhanced. In the same experimental set up, extramedullar hematopoiesis 1s even more strikingly modified. with a dramatic increase in the incidence of CFC (around 40 fold), BFU-E and erythroblasts in the spleen. These modifications attain their maximum 6 days after injection when the bone marrow has returned to normal. The effect of anti-CD3 injection in terms of histamine and IL-6 production by spleen cells is double. namely a complete inhibition after 24h, a return to normal after 4811 and a strong increase after 3-4 days. The initial inhibitory phase is wncwmtenT with the emergence of spleen cells which decrease IL-3induced histamine and IL-6 production by progenitors and abrogate Con A-induced uroliferation of normal soleen cells hv a mechanism which is still uneiplained.
CLONING OF BOVINE STEM CELL FACTOR cDNA. B. Mertens and L.Logan-Henfrey. International Laboratory for Research on Animal Diseases,P.O.Box 30709,Nairobi,KENYA To clone the bovine analogue of the cDNA portion coding for soluble SCF, oligonucleotide primers were designed based on interspecies homologies. Total RNA WFlS isolated from bone marrow mononuclear cells fragments, from Boran cattle. The DNA obtained by RT-PCR, were subcloned and sequenced. The bovine cDNA contains an open reading frame up to the end of the available fragment encoding 174 amino acid residues. Homology with human SCF at the amino acid and nucleotide level is 85 and 90% respectively. Preservation of cysteine residues at position 4, 43, 89 and 139 is thought to result in disulphide bonding identical to that seen in the rodent and primate SCFs. At amino acid position 130 the bovine sequence contains an additional codon, not present in human or rodent SCFs, but present in the canine analogue.
542 I Abstracts
Vol. 6: No. 5 (September
1994: 539-582)
A19 ACTIVATION OF PERIPHERAL BLOOD T CELLS THROUGH LIGANDS OF THE TNF FAMILY. S. Wiley, C. Smith, M. Alderson, and R. Goodwin. lmmunex Corporation, 51 University SL, Seattle WA 98101. Our group is testing the potential of TNF ligand family members lo transduce signals to their host cells independent of their cognate receptors. The TNF ligand family includes several related proteins which can be found on the surface of peripheral blood T (PBT) cells subsequent to stimulation with antiCD3. These proteins include CD30 ligand (CD30L), CD40 ligand (CD40L). Fas ligand, and TNFa. The surface forms of these molecules are type II Vansmembrane proteins with their aminotermini inside of the cell. The amino acid sequences of individual TNF ligand family members' cytoplasmic domains are conserved between species, but are divergent amongst different family members. The percent amino acid conservation of the cytoplasmic domains between the mouse and human forms of CD40L, TNFa dnd CD30L are 82%, 86%, and 61%, respectively. This inter-species sequence conservation strongly suggests some functional role for the cyloplasmic domains of the ligands. We have shown that a monoclonal antibody against human CD30L is able to stimulate PBTs in the presence of anti-CD3. This phenomenon is not a property of CD30L alone, since stimulation using these conditions has been produced with monoclonal antibodies against the CD40L and with a fusion protein consisting of the Fc domain and the extracellular domain of the LTP receptor. These results strongly suggest that some of the TNF family ligands themselves can transduce stimulatory signals lo their host cells, thereby blurring the distinction between receptor and ligand. Further experiments are underway to measure cytokine production and regulation of cell surface markers in PBT cells stimulated with anti-CDBOL and anti-CDS, and lo search for additional biological effects mediated by other TNF ligand family members.
P2 October 4: Lymphokinedcytokines
in Hematopoiesis
A20
A22 Stimulation of Megakaryocytopoiesis Thrombopoiesis by the c-Mpl Frederic
J.
deSauvage
& Dan L.
and ligand
Eaton
The Departments of Molecular Biology & Cardiovascular Research, Genentech Inc., S. San Francisco, CA 94080 Physiological platelet synthesis is thought to require humoral activities known as meg-CSF and thrombopoietin which promote proliferation and maturation of megakaryocytic cells, respectively. Here, we describe the purification and molecular cloning of a meg-CSF/TPO-like protein that is present in plasma of irradiated pigs. This orotein binds to. and activates. c-mol. a member of thk cytokine receptor superfamily: The isolated mpl ligand is a novel cytokine with a unique structure; the amino terminal domain is homologous to erythropoietin and the C-terminal glycosylated domain is unrelated to any known protein. In vitro and in vivo experiments with recombinant mpl ligand indicate that it has both megakaryocyte colony-stimulating activity and thrombopoietin activity, and therefore corresponds to the long sought platelet growth factor, thrombopoietin (TPO).
Effective Cytokine Gene Transfer to Human Bone Marrow Stromal Cells using El Deleted Adenoviral Vectors. R. Foley, T. Braciak, C. Addison, C. Brazolot-Millan, I. Walker, R. Carter, F. Graham, J. Gauldie, McMaster University, Hamilton, Ontario, Canada L8N 325 An adenoviral cytokine gene transfer system was used in human long term bone marrow culture to demonstrate the feasibility of genetically manipulating adherent (stromal) cells to produce functional cytokine at high levels over a period of days. Human bone marrow
samples from healthy
marrow
transplant
donors
were maintained
in
modified Dexter’s long term culture for the generation of an adherent layer representative of the marrow stroma. Adenoviral constructs designed to transfer the gene for P-galactosidase and murine interleukin-6 (IL-4) were used to infect adherent layers. Adenoviral vectors were constructed with cDNA inserted into the El region of the viral genome under the transcriptional control of a human
cytomegalovirus promoter (hCMV). Our results demonstrate effective gene transfer and expression of functional protein at high levels over a period
the range
of at least 10 days in vitro.
of micrograms
response infection)
relationship and cytokine
cytokine
gene transfer
Levels of functional
per millilitre
of culture
IL-6 were in
media.
A dose
between viral concentration (multiplicity of production was seen. We consider adenoviral
in adherent
human marrow
culture
useful in evaluating these relationships and the modulation maturation. (Supported by MRC and NC1 Canada)
potentially of stem cell
A23
A21 EFFECT OF ANTI-CD3 INJECTION ON HEMATOPOIESIS AND IL-3. INDUCED HISTAMWE AND IL-6 PRODUCTION BY PROGENITORS. M. Dy, V. Salaiin , A. Ben Amor, L. Chatenond, C. Le Bousse, E. Schneider. CNRS URA 1461- INSERM U25, Hapita Necker, Paris, INSERM U 268 , Villejnif, FRANCE. Hematopoietic progenitors respond to IL-3 in vitro by producing large nmounts of histamine and IL-6. This has also be demonstrated in viva during antigenic challenge of immunized mice where IL-3 can be detected in the circulation. Herein, we provide evidence for a similar effect of anti-CD3 injection into normal mice, i.e. the appearance of circulating IL-3 accompanied by enhanced activity of histidine decarboxylase. the histamine-forming enzyme, in bone marrow and spleen. In addition, we show that the frequency of CFC and BFU-E in the bone marrow increases substantially during the first days following anti-CD3 administration, while the effect of IL-3 on the ex viva production of histamine and IL-6 by bone marrow cells is also enhanced. In the same experimental set up, extramedullar hematopoiesis 1s even more strikingly modified. with a dramatic increase in the incidence of CFC (around 40 fold), BFU-E and erythroblasts in the spleen. These modifications attain their maximum 6 days after injection when the bone marrow has returned to normal. The effect of anti-CD3 injection in terms of histamine and IL-6 production by spleen cells is double. namely a complete inhibition after 24h, a return to normal after 4811 and a strong increase after 3-4 days. The initial inhibitory phase is wncwmtenT with the emergence of spleen cells which decrease IL-3induced histamine and IL-6 production by progenitors and abrogate Con A-induced uroliferation of normal soleen cells hv a mechanism which is still uneiplained.
CLONING OF BOVINE STEM CELL FACTOR cDNA. B. Mertens and L.Logan-Henfrey. International Laboratory for Research on Animal Diseases,P.O.Box 30709,Nairobi,KENYA To clone the bovine analogue of the cDNA portion coding for soluble SCF, oligonucleotide primers were designed based on interspecies homologies. Total RNA WFlS isolated from bone marrow mononuclear cells fragments, from Boran cattle. The DNA obtained by RT-PCR, were subcloned and sequenced. The bovine cDNA contains an open reading frame up to the end of the available fragment encoding 174 amino acid residues. Homology with human SCF at the amino acid and nucleotide level is 85 and 90% respectively. Preservation of cysteine residues at position 4, 43, 89 and 139 is thought to result in disulphide bonding identical to that seen in the rodent and primate SCFs. At amino acid position 130 the bovine sequence contains an additional codon, not present in human or rodent SCFs, but present in the canine analogue.