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Regulation of macrophage functions by IL-3
106
/ SECOND
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
199
202 REGULATION
OF MACROPHAGE
FUNCTIONS
BY 11-3.
Gyorgy Frsndl, David 1. Bollor, Boston University MedIcal Cantor, Evans Medical Foundaion. Barton, MA 02116
Due to the central role of macrophage (MB) in both T cell activation via antigen presentation and defense against intracellular parasites and tumor cells, it was of interest to understand the process by which T cell cylokines induced MB activation. Here we repart that IL-3 (multi-CSFl has a complex pattern of macrophage activating potential on iunctions related to antigen oresentation. IL-3 induced both IA and IE exoress’on. and was also found to synergize with IFN-y in la induction. IL-3 also displayed a dramatic synergistic interaction with low doses of LPS (0.55ng/ml), though, as was reported earlier (Steeg et al.J.1. 129:2402) LPS at high doses was inhibitory for la induction induced by either IFN-7 or IL-3. The synergism of IL-3 and LPS was not mediated by IL-I, TNF-a, or IL-6 (cytokines induced in MB by LPS). thus suggesting that it might represent a direct effect of IL-3 and LPS on the MB. IL-3 also induced LFA-1 expression, but in distinction to its role in the induction of la, no interaction of IL-3 and LPS was seen during LFA-1 induction. In these regards the activity of IL-3 was similar to that of IFN-y. However, a clear functional distinction between these two lymphokines was found: only IFN-1 was capable of inducing cvtotoxicitv aaainst the murine mastocvtoma P815. IL-3 neither induced killing dir&tl; nor modified the cytotoxic activity induced by IFN-1. IL-3 seems to function in a manner that is both distinct from and synergistic with IFN-y, and (unlike IFN?) is produced by both Thl and Th2 type T helper cells as well as other cell types. Thus, we suggest that IL-3 might play an important role in regulating, in particular, those Me functions which contribute to the antigen presenting functions of MB. Our preliminary results support this hypothesis.
RHEUMATOID ARTHRITIS (RA) SYNOVIAL CELL (SC) GROWTH IN VITRO IS AUGMENTED BY POLYPEPTIDE GROWTH FACTORS (PGFs) AND INTERLEUKIN-1 (IL-1 ).David H.Goddard,Swtt L.Grossman. and Mary E.Moore.Albert Einstein Medical Center,Philadelphia,PA. We have found that RA SCs grow in medium lacking all essential growth factors, suggesting that RA SC growth is driven by the continuous autocrine secretion of at least one growth factor. Several PGFs and cytokines present in RA synovial fluids, could stimulate SC growth. Here we measure the mitogenic effects of PGFs and interleukin-1 (IL-l) alone, and in combination, on established SC lines derived from RA synovial membranes. RA SCs were grown for 14 days in serum-free RPMI, or serum-free RPM1 plus PGFs (TGF-B; or epidermal growth factor, EGF; or platelet-derived growth factor, PDGF; or basic fibroblast growth factor, bFGF), or IL-i, or both. Significant growth was observed in all cell lines compared with day 1 (~(0.01). Compared with growth in RPM1 alone, RA SC growth was greater in RPM1 plus bFGF, and significantly greater in RPM1 plus PDGF, or IL-1 (p
200
203
GM-CSF AND IL 4 REGULATE HISTOCOMF’ATIBILITY ANTIGEN EXPRESSION IN A MANNER DISTINCT FROM INTERFERON-GAMMA. T.L. Gerrard, D.R. Dyer, and H.S. Mostowski. Division of CytZiZe Biology, CBER, FDA, Bethesda, MD 20892
WR NECROSIS FACTOR (INF# ) INFLUENCES Mom M4CROl’KXECUl.ES ACROSS THE PuLMlNARY VASUJIAR -IAL BARRIER. S.E. Goldblm and W.L. Sun. VAMP, Univ Baltimore, MD 21201.
We have observed that GM-CSF can augment expression of class II histocompstibility antigens (HLA-DR) on human monocytes. Monocytes were obtained by elutriation and surface antigens were analyzed by flow cytometry. Thus GM-CSF behaves similarly to interferongamma and IL 4 in the ability to enhance HLA-DR antigen expression on q onocytes, However, neither GM-CSF nor IL 4 were able to enhance the expression of class I histocompatibility antigens. This differs from the action of interferon-gamma which also enhances the expression of class I histocompatibility antigens. We have also examined the effects of these cytokines on a monocytoid cell line, THP-1. IFN-gamma had a similar effect on THP-1 cells to its effect on peripheral blood monocytes, that is, it enhanced the expression of both class I and class II histocompatibility antigens. In contrast, IL 4 and GM-CSF did not enhance the expression of HLA-DR antigens on THP-1 cells, despite their positive action on monocytes. Neither did IL 4 or GM-CSF enhance the expression of class I histocompatibility antigens on THP-1 cells. There may be maturational differences between peripheral blood monocytes and THP-1 cells that account for the differences in susceptibility to signals from IL 4 and GM-CSF.
TNFN has been implicated as a mediator vagfinen~~;~df irj+u”Ty . We studied C bovine
aennn
OF MD, Sch Med,
of pulmonary the effect of tin albumin
(BSA)
across
cultured rlNFw
bovine pulmonary artery endothelial cell monolayers. induced a dose-, time-,lpd temperature-dependent increment in transendothelial C-BSA flux. Only after an incubation of > 4 hr did rTNFp( significantly (p < 0.005) increase trans~ndothelial albunin flux. sure times flNk4 e as brief as 5 min induced significantly (p < 0.005 “p” increased albumin transfer at 6 hr. Although this initial mu endothelial interaction was not temperature dependent, the subsequent barrier dysfunction could only be generated at 37’C. The ?INFM induced changes in endothelial permeability c uld not be ascribed to endothelial cell grow h inhibition 51 (%I-thym.d’ 1 me incorporation) or cytotoxicity ( Chrcmiun release) and was not blocked by prior protein synthesis inhibition. The effects of rTNFo( on endothelial permeability were reversible at 24 hr. lhe effects of rTPJF& on transendothelial f ux of two tracers of similar Ew, 14C-BSA (66,000) and !IH-dextrsn (70,000), were comparable. Thus the change in barrier function was not specific for albumin. Therefore, mo( regulates the movement of macrwnolecules across the pulmonary vascular endothelial barrier and may be operative in the adult respiratory distress syndrane.
201
204
POLYPEPTIDE GROWTH FACTORS REGULATE RHEUMATOID ARTHRITIS (RA) SYNOVIAL CELL ISC) GROWTH IN VITRO. David H. Goddard. Scott L. Grossman. and Mary E. Moore. Albert Einstein Medical Center, Philadelphia, PA We have observed that established cell lines derived from RA, but not osteoarthritis synovial membranes grow in serumfree RPM1 (RPM1 1640 plus non-essential amino-acids, and antibiotics; day 35 vs day 1 p
PRODUCTION OF IL 1, TNF AND IL 6 BY HUNAN OSTEOBLASTS IN VITRO. &aGowen. Amanda Littlewood. David Rictard. Karen ChaDman. David Hushes. Dean Evans an&Graham Russell. Univ. Sheffield Medical School,Sheffield SlO 2RX and Bath Institute for Rheumatic Diseases, Bath BAl lH0, UK. Cytokines are known to regulate both bone formation and bone resorption. Molecules such as IL 1, IL 6 and TNF have been implicated in diseases involving bone loss, for example rheumatoid arthritis, myeloma and osteoporosis. In these situations the source of the factors has been thought to be cells associated with the pathology. We have postulated that they may also control normal bone rencdelling and that bone cells themselves may be capable of their production. Using specific bioassays and neutralisation of activity with specific antibodies we have shown that human trabecular bone cells expressing an osteoblastic phenotype are capable of producing all three cytokines. IL 1 was released (lo-‘-10-‘=%) after induction with LPS or TNF. TNF was released at low levels by unstimulated cells but at higher levels (lO-**-lO-*“M) after induction with LPS, IL 1 or CM-CSF, but not IL 6. IL 6 was released under basal conditions (150018000 U/ml) and this was also enhanced by stimulation with IL 1, LPS and TNF up to 100000 U/ml (approx 10--N). The release of these factors was not affected by the osteotropic hormones 1,25-dihydroxyvitamin DI or parathyroid hormone. These results suggest that local production of such factors could represent paracrine or autocrine control mechanisms of the highly localised process of bone remodelling.
3T3 fibroblasts, effect data
TGF-B
on cell
specifically show
factor, of such stimulus
growth,
cytotoxic that
which
antibody
(10 or 100 &ml
indicating
to cells
that
RA SCs in culture
could
be TGF-B.
secrete
Continuous
a growth factor could provide for RA SC growth in vitro.
IgG) had no
it was not. non-
in culture.
Taken
together
at least
autocrine an important
these
one growth
secretion mitogenic
/ SECOND
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
199
202 REGULATION
OF MACROPHAGE
FUNCTIONS
BY 11-3.
Gyorgy Frsndl, David 1. Bollor, Boston University MedIcal Cantor, Evans Medical Foundaion. Barton, MA 02116
Due to the central role of macrophage (MB) in both T cell activation via antigen presentation and defense against intracellular parasites and tumor cells, it was of interest to understand the process by which T cell cylokines induced MB activation. Here we repart that IL-3 (multi-CSFl has a complex pattern of macrophage activating potential on iunctions related to antigen oresentation. IL-3 induced both IA and IE exoress’on. and was also found to synergize with IFN-y in la induction. IL-3 also displayed a dramatic synergistic interaction with low doses of LPS (0.55ng/ml), though, as was reported earlier (Steeg et al.J.1. 129:2402) LPS at high doses was inhibitory for la induction induced by either IFN-7 or IL-3. The synergism of IL-3 and LPS was not mediated by IL-I, TNF-a, or IL-6 (cytokines induced in MB by LPS). thus suggesting that it might represent a direct effect of IL-3 and LPS on the MB. IL-3 also induced LFA-1 expression, but in distinction to its role in the induction of la, no interaction of IL-3 and LPS was seen during LFA-1 induction. In these regards the activity of IL-3 was similar to that of IFN-y. However, a clear functional distinction between these two lymphokines was found: only IFN-1 was capable of inducing cvtotoxicitv aaainst the murine mastocvtoma P815. IL-3 neither induced killing dir&tl; nor modified the cytotoxic activity induced by IFN-1. IL-3 seems to function in a manner that is both distinct from and synergistic with IFN-y, and (unlike IFN?) is produced by both Thl and Th2 type T helper cells as well as other cell types. Thus, we suggest that IL-3 might play an important role in regulating, in particular, those Me functions which contribute to the antigen presenting functions of MB. Our preliminary results support this hypothesis.
RHEUMATOID ARTHRITIS (RA) SYNOVIAL CELL (SC) GROWTH IN VITRO IS AUGMENTED BY POLYPEPTIDE GROWTH FACTORS (PGFs) AND INTERLEUKIN-1 (IL-1 ).David H.Goddard,Swtt L.Grossman. and Mary E.Moore.Albert Einstein Medical Center,Philadelphia,PA. We have found that RA SCs grow in medium lacking all essential growth factors, suggesting that RA SC growth is driven by the continuous autocrine secretion of at least one growth factor. Several PGFs and cytokines present in RA synovial fluids, could stimulate SC growth. Here we measure the mitogenic effects of PGFs and interleukin-1 (IL-l) alone, and in combination, on established SC lines derived from RA synovial membranes. RA SCs were grown for 14 days in serum-free RPMI, or serum-free RPM1 plus PGFs (TGF-B; or epidermal growth factor, EGF; or platelet-derived growth factor, PDGF; or basic fibroblast growth factor, bFGF), or IL-i, or both. Significant growth was observed in all cell lines compared with day 1 (~(0.01). Compared with growth in RPM1 alone, RA SC growth was greater in RPM1 plus bFGF, and significantly greater in RPM1 plus PDGF, or IL-1 (p
200
203
GM-CSF AND IL 4 REGULATE HISTOCOMF’ATIBILITY ANTIGEN EXPRESSION IN A MANNER DISTINCT FROM INTERFERON-GAMMA. T.L. Gerrard, D.R. Dyer, and H.S. Mostowski. Division of CytZiZe Biology, CBER, FDA, Bethesda, MD 20892
WR NECROSIS FACTOR (INF# ) INFLUENCES Mom M4CROl’KXECUl.ES ACROSS THE PuLMlNARY VASUJIAR -IAL BARRIER. S.E. Goldblm and W.L. Sun. VAMP, Univ Baltimore, MD 21201.
We have observed that GM-CSF can augment expression of class II histocompstibility antigens (HLA-DR) on human monocytes. Monocytes were obtained by elutriation and surface antigens were analyzed by flow cytometry. Thus GM-CSF behaves similarly to interferongamma and IL 4 in the ability to enhance HLA-DR antigen expression on q onocytes, However, neither GM-CSF nor IL 4 were able to enhance the expression of class I histocompatibility antigens. This differs from the action of interferon-gamma which also enhances the expression of class I histocompatibility antigens. We have also examined the effects of these cytokines on a monocytoid cell line, THP-1. IFN-gamma had a similar effect on THP-1 cells to its effect on peripheral blood monocytes, that is, it enhanced the expression of both class I and class II histocompatibility antigens. In contrast, IL 4 and GM-CSF did not enhance the expression of HLA-DR antigens on THP-1 cells, despite their positive action on monocytes. Neither did IL 4 or GM-CSF enhance the expression of class I histocompatibility antigens on THP-1 cells. There may be maturational differences between peripheral blood monocytes and THP-1 cells that account for the differences in susceptibility to signals from IL 4 and GM-CSF.
TNFN has been implicated as a mediator vagfinen~~;~df irj+u”Ty . We studied C bovine
aennn
OF MD, Sch Med,
of pulmonary the effect of tin albumin
(BSA)
across
cultured rlNFw
bovine pulmonary artery endothelial cell monolayers. induced a dose-, time-,lpd temperature-dependent increment in transendothelial C-BSA flux. Only after an incubation of > 4 hr did rTNFp( significantly (p < 0.005) increase trans~ndothelial albunin flux. sure times flNk4 e as brief as 5 min induced significantly (p < 0.005 “p” increased albumin transfer at 6 hr. Although this initial mu endothelial interaction was not temperature dependent, the subsequent barrier dysfunction could only be generated at 37’C. The ?INFM induced changes in endothelial permeability c uld not be ascribed to endothelial cell grow h inhibition 51 (%I-thym.d’ 1 me incorporation) or cytotoxicity ( Chrcmiun release) and was not blocked by prior protein synthesis inhibition. The effects of rTNFo( on endothelial permeability were reversible at 24 hr. lhe effects of rTPJF& on transendothelial f ux of two tracers of similar Ew, 14C-BSA (66,000) and !IH-dextrsn (70,000), were comparable. Thus the change in barrier function was not specific for albumin. Therefore, mo( regulates the movement of macrwnolecules across the pulmonary vascular endothelial barrier and may be operative in the adult respiratory distress syndrane.
201
204
POLYPEPTIDE GROWTH FACTORS REGULATE RHEUMATOID ARTHRITIS (RA) SYNOVIAL CELL ISC) GROWTH IN VITRO. David H. Goddard. Scott L. Grossman. and Mary E. Moore. Albert Einstein Medical Center, Philadelphia, PA We have observed that established cell lines derived from RA, but not osteoarthritis synovial membranes grow in serumfree RPM1 (RPM1 1640 plus non-essential amino-acids, and antibiotics; day 35 vs day 1 p
PRODUCTION OF IL 1, TNF AND IL 6 BY HUNAN OSTEOBLASTS IN VITRO. &aGowen. Amanda Littlewood. David Rictard. Karen ChaDman. David Hushes. Dean Evans an&Graham Russell. Univ. Sheffield Medical School,Sheffield SlO 2RX and Bath Institute for Rheumatic Diseases, Bath BAl lH0, UK. Cytokines are known to regulate both bone formation and bone resorption. Molecules such as IL 1, IL 6 and TNF have been implicated in diseases involving bone loss, for example rheumatoid arthritis, myeloma and osteoporosis. In these situations the source of the factors has been thought to be cells associated with the pathology. We have postulated that they may also control normal bone rencdelling and that bone cells themselves may be capable of their production. Using specific bioassays and neutralisation of activity with specific antibodies we have shown that human trabecular bone cells expressing an osteoblastic phenotype are capable of producing all three cytokines. IL 1 was released (lo-‘-10-‘=%) after induction with LPS or TNF. TNF was released at low levels by unstimulated cells but at higher levels (lO-**-lO-*“M) after induction with LPS, IL 1 or CM-CSF, but not IL 6. IL 6 was released under basal conditions (150018000 U/ml) and this was also enhanced by stimulation with IL 1, LPS and TNF up to 100000 U/ml (approx 10--N). The release of these factors was not affected by the osteotropic hormones 1,25-dihydroxyvitamin DI or parathyroid hormone. These results suggest that local production of such factors could represent paracrine or autocrine control mechanisms of the highly localised process of bone remodelling.
3T3 fibroblasts, effect data
TGF-B
on cell
specifically show
factor, of such stimulus
growth,
cytotoxic that
which
antibody
(10 or 100 &ml
indicating
to cells
that
RA SCs in culture
could
be TGF-B.
secrete
Continuous
a growth factor could provide for RA SC growth in vitro.
IgG) had no
it was not. non-
in culture.
Taken
together
at least
autocrine an important
these
one growth
secretion mitogenic