Several pathways for bFGF endocytosis in abae cells
1
Biol. Cell, Vol 76, 1992 223 TIIE ROLE OF EPITOPES IN SELECTIVE T-CELL ACTIVATION. F..,~taquierJ~r~51ne*. Boutillon Chdstophe$, Gras-Masse I-MI4ne$. I)aprez Benoit$. Capron Andr,~*, Tartar AndrOS, and Auriault Claude*. * Cenlre d'lmmunologie des maladies tran.slnissibles et allergiques,Unil~mixte INSERM 167-CNRS 624, Institul Pasteur Lille; $ Chimiedes biomol&:ules, URA-CNRS1309, InsfitutPasteur lille. CD4 T-cells play a pivotol role in the immune response to protein antigens. Attention has focused on the responsability of this cell population for the heterogeneity of immune response, mediated predominantly by the secretion of cytokines. Indeed, these cells are functionally distinguishable regarding their lymphokine repertoire: "Thl" cells produce IL-2 and IFNy whereas "Th2" cells secrete IL-4, IL-5 and IL-6. Importantly. determination of tim factors responsible for selectively activating these two T-cell subsets is crucial. We have studied the immune response against a combinative peptide, called a mixotope, a mixture of 7.5 1135related peptides representing different natural variants of the V3 loop of the env HIV-I protein. Our results show that such immunogen induce a high level of T-cell proliferation irrespective of the haplotype tested and produce a wide range of lymphokines. In contrast, using some individual V3 sequences as a stimulants (V3 RF, SF2, BRU, MN, and ELI) we were able to induce the selective production of lymphokines in the absence of detectable proliferative response. This production varied according to the MHC haplotypes invoh,ed in the antigenic presentation. Thus, specific antigen stimulation could dissociate T-cell proliferation from lymphokine production. The activation of CD4 T-ceils requires the fgrmation of a trimolccular complex between an antigenic peptide, MHC molecule and the T cell receptor (TCR). These results suggest that differences in the peptide-MHC interaction among the V3 variants presented, leads to distinct regulatory signals at the T-cell level. Such difference might be transmitted by diverse "second messagers" leading to distinct lymphokine expression. The determination of the nature of these messagers and their role at the ,uclear level remain to be clarified. IMMUNOREACq'IVITY OF INSULIN (B), GLUCAGON (A), SOMATOS'FATIN (D) AND PANCREATIC POLYPEIrFIDE (F) CELLS IN THE PANCREAS OF "File GARDEN DORMOUSE DURING EXPERIMENTAL AROUSAL : 1MPLICATION OF CATECHOI..AMINES GAILLARD Genevieve (1), CORTIE Chautal (2), IIOLLANDE Etienne (3) and AMBID Louis (1) (1) Laboratoire des rdgulatiolL~ des m~tabolismes et nutrition, (2) Laboratoire de digestion et nutrition, (3) Laboratoire de biologie celhdaire. Universitd Paul Sabatier, Toulouse (France) Previous studies on the mobilization of hepatic carbohydrates during arousal of the hibernating garden dormouse have indicated that different physiological functions became acti~,ated at characterisdc temperatures. In this study, the influence of body temperature on the reactivation of the endocrine pa,mreas was investigated during spontaneous fluctuations in thermogenesis. Insulin (13), glucagon (A), somatostatin (D) and pancreatic polypeptide (F) cells were visualized inununocytochemically. Plasma glucose, catecholamines (CA), dopamine fi-hydroxylase (Dill-l) and insulin levels were measured dr,ring hypothermy and at different stages of arousal. During the lethargic period (60C), all endocrine pancreatic cells were weakly itmnunoreactive. No structural or metabolic alterations were noted as body temperature rose from 6°C to 12°C. At 19"C, there was a riseliq immunoreactivity of A cells, although glucose was low (0.37 rag.l" ), while levels of CA were three-fold the basal hibernating level. Plasma insulin levels rose slightly between 19°C and 28°C, reaching a peak between 29°C and 36"C. Over the latter period, B and D cells showed a marked positivity along with a five-fold increase in CA levels. Plas,na glucose was normal between 29"C and 36"C, reaching a peak during honaeothermy, concomitant with high levels of DBII and CA. Maxinmm immunostaining of F cells was observed during et, thermy. In conclusion, peripheral sympathetic activity was thought to be responsible for the sequential reactivation of pancreatic cells during arousal of the garden dormouse. SEVERAL PATHWAYS FOR bFGF ENDOCYTOSIS IN ABAE CELLS 0LEIZES Pierre-Emmanuel, NOAILLAC-DEPEYRE Jacqueline, DUPONT Marie-Ange, GAS Nicole Institut de Biologic Cellulaire el de Gdndtique, 118 route de Narbonne, 31062 Toulouse cEdex. Different kinds of receptors have been found to mediate basic fibroblast growth factor (bFGF) action in several cell lines. One can distinguish high affinity receptors, presumably located on plasma membrane, and low affinity receptors identified as plasma membrane and extracellular matrix (ECM) heparan-sulfate (1). By electron microscopy, we have examined the pathways of bFGF endocytosis in adult bovine aortic endothelial (ABAE) cells, which are bFGF-responsive for growth and differenciation. Recombinant bovine bFGF (146 a.a.) was conjugated to horse radish peroxydase, or to 10 nm gold particles. Both probes were found to recognize specifically bFGF binding sites. However, the conjugates were not mitogenic. Direct visualization of labelled bFGF entry showed different ways of endocytosis. On apical surface, internalization occured by coated pits, non-coated pits, and caveolae. On basal surface, ECM exhibited a strong labelling. Abgve highly labelled ECM parts, we could observe smoo{h vesicles piching off, filled with matrix linked bFGF. These results suggest that ABAE cells internalize exogenous bFGF by different pathways on apical plasma membrane, and can also internalize ECM bound exogenous bFGF by endocytosis on basal surface. (l) Klagsbrun M and Baird A. (1991). Cell, 67, 229-231. Nuclear accumulation of Vasculotropin/VEGF. L. Gouzi, G. Bouche, J. Plouet, Laboratoire de Biologie Mol4culaire des Eucaryotes, 31062, Toulouse. Vasculotropin (VAS), also called Vascular Endothelial Growth Factor, is an angiogenic cytokine purified from the conditioned medium of various normal and tumoral cells. VAS stimulates the proliferation of endothelial cells cultured fgom aorta (BAE) and not from cornea (BCE) although it binds to the same extent to both cells. We looked whether this discrepancy might be related to a difference in VAS nuclear accumulation. Subconfluent cells were exposed during 2 hours at 37°C in presence of 1 ng/ml of iodinated VAS, the nucleus were fractionated and the nuclear content was analysed. 10% and 13% of the VAS bound was associated with the nucleus of BCE and BAE respectively. The presence of a 300 molar excess of VEGF or its inactivation reduced both the cytoplasmic and the nuclear contents by 80%. Similar results were obtained with native and he~t inactivated bFGF. These preliminary results suggest that the nuclear accumulation of VAS might be not related to its mitogenic function.
Transcript of Several pathways for bFGF endocytosis in abae cells
Biol. Cell, Vol 76, 1992 223
TIIE ROLE OF EPITOPES IN SELECTIVE T-CELL ACTIVATION. F..,~taquier J~r~51ne*. Boutillon Chdstophe$, Gras-Masse I-MI4ne$. I)aprez Benoit$. Capron Andr ,~*, Tartar AndrOS, and Auriault Claude*. * Cenlre d'lmmunologie des maladies tran.slnissibles et allergiques, Unil~ mixte INSERM 167-CNRS 624, Institul Pasteur Lille; $ Chimie des biomol&:ules, URA-CNRS 1309, Insfitut Pasteur lille.
CD4 T-cells play a pivotol role in the immune response to protein antigens.
Attention has focused on the responsability of this cell population for the
heterogeneity of immune response, mediated predominantly by the secretion of
cytokines. Indeed, these cells are functionally distinguishable regarding their
lymphokine repertoire: "Thl" cells produce IL-2 and IFNy whereas "Th2" cells
secrete IL-4, IL-5 and IL-6. Importantly. determination of tim factors
responsible for selectively activating these two T-cell subsets is crucial.
We have studied the immune response against a combinative peptide, called a
mixotope, a mixture of 7.5 1135 related peptides representing different natural
variants of the V3 loop of the env HIV-I protein. Our results show that such
immunogen induce a high level of T-cell proliferation irrespective of the
haplotype tested and produce a wide range of lymphokines.
In contrast, using some individual V3 sequences as a stimulants (V3 RF,
SF2, BRU, MN, and ELI) we were able to induce the selective production of
lymphokines in the absence of detectable proliferative response. This production
varied according to the MHC haplotypes invoh,ed in the antigenic presentation.
Thus, specific antigen stimulation could dissociate T-cell proliferation from
lymphokine production.
The activation of CD4 T-ceils requires the fgrmation of a trimolccular
complex between an antigenic peptide, MHC molecule and the T cell receptor
(TCR). These results suggest that differences in the peptide-MHC interaction
among the V3 variants presented, leads to distinct regulatory signals at the T-cell
level. Such difference might be transmitted by diverse "second messagers"
leading to distinct lymphokine expression. The determination of the nature of
these messagers and their role at the ,uclear level remain to be clarified.
IMMUNOREACq'IVITY OF INSULIN (B), GLUCAGON (A), SOMATOS'FATIN (D) AND PANCREATIC POLYPEIrFIDE (F) CELLS IN THE PANCREAS OF "File GARDEN DORMOUSE DURING EXPERIMENTAL AROUSAL : 1MPLICATION OF CATECHOI..AMINES
GAILLARD Genevieve (1), CORTIE Chautal (2), IIOLLANDE Etienne (3) and AMBID Louis (1) (1) Laboratoire des rdgulatiolL~ des m~tabolismes et nutrition, (2) Laboratoire de digestion et nutrition, (3) Laboratoire de biologie celhdaire. Universitd Paul Sabatier, Toulouse (France)
Previous studies on the mobilization of hepatic carbohydrates during arousal of the hibernating garden dormouse have indicated that different physiological functions became acti~,ated at characterisdc temperatures.
In this study, the influence of body temperature on the reactivation of the endocrine pa,mreas was investigated during spontaneous fluctuations in thermogenesis.
Insulin (13), glucagon (A), somatostatin (D) and pancreatic polypeptide (F) cells were visualized inununocytochemically. Plasma glucose, catecholamines (CA), dopamine fi-hydroxylase (Dill-l) and insulin levels were measured dr,ring hypothermy and at different stages of arousal.
During the lethargic period (60C), all endocrine pancreatic cells were weakly itmnunoreactive. No structural or metabolic alterations were noted as body temperature rose from 6°C to 12°C. At 19"C, there was a riseliq immunoreactivity of A cells, although glucose was low (0.37 rag.l" ), while levels of CA were three-fold the basal hibernating level. Plasma insulin levels rose slightly between 19°C and 28°C, reaching a peak between 29°C and 36"C. Over the latter period, B and D cells showed a marked positivity along with a five-fold increase in CA levels. Plas,na glucose was normal between 29"C and 36"C, reaching a peak during honaeothermy, concomitant with high levels of DBII and CA. Maxinmm immunostaining of F cells was observed during et, thermy.
In conclusion, peripheral sympathetic activity was thought to be responsible for the sequential reactivation of pancreatic cells during arousal of the garden dormouse.
S E V E R A L P A T H W A Y S FOR b F G F E N D O C Y T O S I S IN A B A E C E L L S 0LEIZES Pierre-Emmanuel, NOAILLAC-DEPEYRE Jacqueline, DUPONT Marie-Ange, GAS Nicole Institut de Biologic Cellulaire el de Gdndtique, 118 route de Narbonne, 31062 Toulouse cEdex.
Different kinds of receptors have been found to mediate basic fibroblast growth factor (bFGF) action in several cell l ines . One can d i s t ingu ish high a f f in i ty r ecep to r s , presumably located on plasma membrane, and low affinity r e c e p t o r s i d e n t i f i e d as p l a s m a m e m b r a n e and extracellular matrix (ECM) heparan-sulfate (1). By electron microscopy, we have examined the pathways of bFGF endocytosis in adult bovine aortic endothelial (ABAE) cells, which are bFGF-responsive for growth and differenciation. Recombinant bovine bFGF (146 a.a.) was conjugated to horse radish peroxydase, or to 10 nm gold particles. Both probes were found to recognize specifically bFGF binding sites. However , the conjugates were not mitogenic. Direct visualization of labelled bFGF entry showed different ways of endocytosis . On apical surface, internalization occured
b y coated pits, non-coated pits, and caveolae. On basal surface, ECM exhibited a strong labelling. Abgve highly labelled ECM parts, we could observe smoo{h vesic les piching off, filled with matrix linked bFGF. These results suggest that ABAE cells internalize exogenous bFGF by different pathways on apical plasma membrane, and can also internalize ECM bound exogenous bFGF by endocytosis on basal surface.
( l) Klagsbrun M and Baird A. (1991). Cell, 67, 229-231.
Nuclear accumulation of Vasculotropin/VEGF.
L. Gouzi, G. Bouche, J. Plouet, Laboratoire de Biologie Mol4culaire des Eucaryotes, 31062, Toulouse.
Vasculotropin (VAS), also called Vascular
Endothelial Growth Factor, is an angiogenic cytokine
purified from the conditioned medium of various
normal and tumoral cells. VAS stimulates the
proliferation of endothelial cells cultured fgom
aorta (BAE) and not from cornea (BCE) although it
binds to the same extent to both cells. We looked
whether this discrepancy might be related to a
difference in VAS nuclear accumulation.
Subconfluent cells were exposed during 2 hours
at 37°C in presence of 1 ng/ml of iodinated VAS, the
nucleus were fractionated and the nuclear content
was analysed. 10% and 13% of the VAS bound was
associated with the nucleus of BCE and BAE
respectively. The presence of a 300 molar excess of
VEGF or its inactivation reduced both the
cytoplasmic and the nuclear contents by 80%. Similar
results were obtained with native and he~t
inactivated bFGF.
These preliminary results suggest that the
nuclear accumulation of VAS might be not related to
its mitogenic function.
