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Differential effects of concanavalin A, serum and cytokines on proteoglycan elaboration and DNA synthesis by mononuclear cells from human peripheral blood
Cell Biology
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Reports,
721
Vol. 15, No. 8, 7 99 1
DIFFERFNTIAL EFFECTS OF CONCANAVALIN A, SERUM CYTOKINES ON PROTEOGLYCAN ELABORATION AND DNA SYNTHESIS MONONUCLEAR CELLS FROM HUMAN PERIPHERAL BLOOD Tassos
Anastassiades*, Anne
Departments University,
of Medicine Kingston,
Ravi Chopra, Wood
Mary
and Biochemistry, Ontario, Canada
*Corresponding
Elliott
AND BY and
Queen's K7L 3N6
Author
ABSTRACT
Human blood derived mononuclear cell (MC) cultures required concanavalin A (Con A) stimulation to synthesize and secrete into the medium high levels of a proteaseresistant proteoglycan (PG) containing predominantly chondroitin sulfate (CS), which was elaborated largely by T-cells in culture. PG and DNA synthesis were studied in MC cultures in the absence and presence of Con A as well as serum and some biologically active polypeptide factors. In the presence of Con A, stimulation of PG synthesis was substantially greater in T-cell enriched cultures than in also B-cell enriched cultures. DNA synthesis was stimulated in the presence of Con A. This stimulation was concentration-dependent, but required the presence of serum for additional responses. DNA and cell proliferation were stimulated by interleukin-2 (IL-2), but PG production was not stimulated by conditioned media, IL-l, IL-2, ILgrowth factor-8 (TGF-8). Our ressults 3, or transforming indicate that the elaboration of PG from T-cells of human MC is independent of the effects of regulatory peptides on cell proliferation and DNA synthesis. INTRODUCTION
PG are anionic glycoconjugates which are synthesized primarily by the differentiated cells of the connective tissues and may serve as organizers of the extracellular matrix (Muir, 1983). However, blood leucocytes, including the polymorphonuclear leucocytes, monocytes, and natural killer cells also elaborate PG that are stored in secretory granules (Stevens et al., 1988). In many chronic inflammatory states, such as rheumatoid arthritis, MC accumulate in the affected connective tissues where they are thought to undergo antigenic stimulation. We had reported that PG synthesis was detectable only at a very low level in MC cultures from human blood, but these cultures could be stimulated by Con A to secrete very high the major anionic levels of a CS PG, which represented glycoconjugate elaborated by these cells (Anastassiades et 0309-I
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al., 1985, 1987). This PG is protease-resistant, requires the presence of serum for maximal response and appears in the medium relatively late in the culture period, generally between 3-9 days (Chopra et al., 1991). This late appearance of the MC PG is in contrast to the pattern of elaboration of a number of lymphokines, including IL-2 produced by T cell clones, which achieved maximal levels in the medium by 24-48 h after stimulation (Otten et al., 1985), and fibroblast growth suppressive activity from Con A-stimulated human lymphocytes which was detected at significant levels by 17 h of culture (Anastassiades and Wood, 1983). We have undertaken a systematic study of conditions affecting PG elaboration by human MC from the blood and, in this paper, we report on the effects of the addition of IL-l, IL-2 on PG and DNA synthesis in this system, as well as the effects of factors known to stimulate PG synthesis of connective tissue cells, such as TGF-8 and "Matrigenin" activity (Anastassiades et al., 1987). MATERIALS
AND METHODS
Isolation and separation of MC: MC (or l~whole~~ MC) were isolated from the peripheral blood of healthy human volunteers by a Ficoll-Hypaque gradient procedure (Anastassiades and Wood, 1981, 1983) and separated into a T-cell enriched population and a B-cell enriched population on a nylon wool column, as previously described (Handwerger and Schwartz, 1974; Chopra et al., 1991). The individually, or separated populations were cultured llreconstitutedVV in the proportions of B and T cells MC were also present in the "Whole" MC population. separated on the basis of their adherance to plastic into adherent (Ad) and non-adherent (NAd) populations. MC were suspended in appropriate medium, incubated in plastic were removed, dishes for 2h or 24 h, the NAd cells centrifuged, suspended in the appropriate incubation medium and seeded in 2 ml medium/well. Cultures referred to as *VrecombinedV' had the resuspended cells added back to the Ad cells. 1.
2. Preparation of conditioned media: NAd, Ad, recombined or unseparated MC were incubated in serum-free or serumcontaining medium (10% fetal bovine serum, FBS) for 1 to incubation, the supernatants were removed, 6 days. After the cells spun down, the supernatants (conditioned media) were filtered (Nalgene syringe filters, 0.2 /.J) and stored at 4°C. 3.
Metabolic
labelling
*VWholelV MC, separated MC were incubated in
and
treatments
MC populations, 35 mm' plastic
of
cell
cultures:
or "reconstituted" wells in CMRL 1969
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medium containing 10% FBS and the radioactive precursors of PG (1.25 /Xi/ml 'H-glucosamine, 30-60 Ci/mmole and 2.0 /Xi/ml "S-sulfate, lo-100 mCi/mmole; DuPont-NEN) added at the start of incubation period (continuous labelling protocol). For the pulse labelling protocol 2.5 pCi/ml 'Hglucosamine and 4 pCi/ml "S-sulfate were added 18 h prior to termination of incubation. Incubations were performed in the absence or presence of 12.5 pg/ml Con A, or conditioned media, or substances listed below, added at the begining of the incubation period at the concentrations indicated under Results: IL-l and IL-2 (Intermedico Co.); TGF-B (R and D systems): phorbol 12myristate 13 acetate and methylumbelliferyl-B-D-xyloside (Sigma Chemical Co.): Hydrocortisone sodium succinate (Upjohn Co.). 4. Counting of Ad and NAd MC cell populations: At the completion of incubations, the NAd cell pellets obtained as described above, were suspended in Hematall isotonic diluent (Fisher Scientific) and counted (Coulter Counter Model 3F). The Ad cells at the bottom of the dishes were incubated with 1.5 ml trypsin (0.25% - Gibco) at 37OC until most cells were detached, the suspension was transferred to a tube containing 2 ml medium (10% FBS) and the procedure was repeated, ensuring that no cells remained in the dish as determined by microscopy. 6. into Assay of radioactive incorporation glycosaminoglycans (GAG): After incubation radioactive GAG were isolated from the media and cells following papain digestion, precipitation by cetyl pyridinium chloride, and separation by cellulose acetate electrophoresis as previously described (Hronowski and Anastassiades, 1979, 1980) and the radioactivity incorporated in to the isolated GAG was determined. 7. Incorporation of C3H]-thymidine: MC were suspended in CMRL 1969 medium containing 10% FBS, or in the serum-free medium and seeded into 96-well plates. Cells were treated with varying concentrations of Con A and incubated for 1 to 9 days, followed by pulse labelling with 0.5 @Y/well of [methyl-'HI-thymidine (20 Ci/mmol, DuPont-NEN) for 18 hrs before termination of the culture, the cells were semi-automatic harvester the collected by a and incorporated radioactivity determined as previously described (Anastassiades and Wood, 1983; Anastassiades et al., 1987). RESULTS by MC populations 1. PG synthesis cells: Both T-cell and B-cell
enriched enriched
in T- and Bpopulations
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Vol. 15, No. 8, 1991
isolated by the nylon wool column technique and cultured separately, as well as "Whole" MC which not stimulated with Con A (NS), produced ;ery littlewe;: that could be detected in the medium of the cultures (Fig. 1). However, when Con A was added there were large increases in the accumulation of radiolabelled PG in the medium of cultures of 8'wholeVV, T-cell-enriched and V1reconstituted'V populations, while the B-cell-enriched population showed only a modest increase in the PG accumulation. 2.
Effects
of
serum
and
Con
A on
DNA
synthesis
by
MC:
Initial experiments indicated that Ad populations elaborated very little GAG, while NAd populations incorporated as much, or more, radiocativity from 3Hglucosamine and ?3-sulfate into medium CS as the lUreconstitutedl' population. Further, polypeptide "mediators", such as IL-l, which were presumably released from the adherent MC during the early part of the culture period, did not play an important role in the elaboration of PG appearing in the medium in this system (unpublished results). This notion as well as the relationship of the stimulation of DNA synthesis and cell replication in Con *-8 ‘0
Figure 1. Radiolabelled GAG produced by B-Cell-enriched T-Cell-enriched (T), "ReconstitutedVV (B+T) and (B), l'Wholel' (W) MC populations in culture. All cultures contained 2~10~ cells per dish and were incubated for 7 days in continuous culture in medium containing 10% FBS and the radioactive precursors of PG. Replicate (at least 4) culture plates were either not stimulated (NS) or
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Vol. 15, No. 8, 199 1
725
stimulated with 12.5 pg/ml Con A (Con A). Only 3H counts incorporated into the isolated and separated GAG are shown for clarity as the "S counts followed a similar pattern. The bars indicate standard errors from the mean.
6.
2
4 TIME
6 IN CULTURE
8 ( DAYS
IO 1
8.
2
4 TIME
6 IN CULTURE
8 ( DAYS
IO 1
2. The relation of Con A concentration to jHthymidine incorporation by MC. Con A was added at the indicated concentrations (as pg/ml culture medium) at the begining of the culture period and the cultures were incubated either in 10% FBS-containing medium (A) or in serum-free conditions (B). -_----------------------------------------------------to the accumulation of medium A-stimulated MC cultures, were further PG (measured as radiolabelled GAG) evaluated. DNA synthesis was studied in the MC system, using a standard pulse labelling technique with 'Hthymidine, under both serum-containing and serum-free culture conditions (Fig. 2). For cultures incubated in 2 A) increasing medium containing 10% FBS (Fig. concentrations of Con A led to increased incorporation of generally similar to that 'H-thymidine, in a pattern previously observed for GAG synthesis (Chopra et al., The optimal Con A concentration required to 1991). stimulate incorporation of 'H-thymidine was less for serumfree media (Fig. 2B) than for serum-containing media. 'H-
Figure
726
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Vol. 15, No. 8, 199 7
Figure 3. Effects of biologically active peptides on cell numbers, DNA synthesis and GAG synthesis. Ad, NAd cell numbers (A), incorporation of 'H-thymidine into cellular DNA (B) and 'H incorporation from 3H-glucosamine into chodroitin sulfate, CS, and hyaluronic acid, HA (C) were assessed after 7 days of incubation of the MC cultures (pulse labelling protocols). At the beginning of the incubation period Con A, or, a peptide factor was added to the cultures and the additions (or controls) are indicated under the bar graphs in panel C and apply also to A and B. Designations and concentrations are: Con A, 12.5 pg/ml Con A; NS, non-stimulated (controls); 11,(l), IL-l at 0.5 u/ml; at 1.5 u/ml; 11,(l), IL-2 at 20 u/ml; 11,(h), IL(h) r IL-l IL-2 at 100 u/ml; 11,+,(l), IL-1 at 0.5 u/ml and IL-2 at 20 u/ml; IL-1 at 1.5 u/ml and IL-2 at 100 u /ml; 11,+,(h), TGF-I3 at 2 rig/ml; TGF(h), TGF-A at 5 rig/ml. 'J='(l), --------------------------------------------------------thymidine responses to Con A were well developed by 4 days in cultures that contained serum, but were diminished and detectable only at 4 days (at 1.25 I.cg/ml of Con A) if serum was ommitted from the culture (Fig. 2A and 2B).
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3. Comparison substances on
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of effects GAG elaboration
of
some biologically
active
As mentioned in the Introduction, newly synthesized GAG appears in the medium relatively late in the culture period, after Con A stimulation (Chopra et al., 1991). In order to examine this observation further cross-feeding experiments with "conditioned" media were initially carried out. However, the addition of supernatants from Con-A stimulated or non-stimulated Ad, NAd or ~~Whole~~MC cultures to non-stimulated Ad, NAd or "whole" MC cultures failed to stimulate the appearance of radiolabelled GAG in the media of these cultures. Also, there was no additional stimulation of GAG elaboration by Con A when conditioned media (from Ad, NAd or "whole" cultures, both stimulated and non-stimulated with Con A) were used to supplement NAd populations. and
DNA
synthesis:
In order to study the relationship of GAG elaboration to DNA synthesis selected biologically active peptides, that would be expected to be elaborated by MC cultures, were then added, either individually or in combinations, to the culture system (Fig. 3). The addition of the higher concentration of IL-2 (100 u/ml), with or without IL-l, resulted in large increases in the total numbers of cells, primarily the Ad population (Fig. 3 A), and both the high and low concentrations of IL-2 stimulated incorporation of 'H-thymidine into DNA (Fig. 3 B). TGF-A (at concentrations shown to stimulate growth and PG synthesis of fibroblastic cultures, see Discussion) had no effect on growth, DNA synthesis or GAG synthesis in this system. Neither the addition of IL-l nor IL-2 had any significant effect on incorporation of 'H or '?S into the isolated GAG (Fig. 3 C), in spite of the stimulatory effects of these factors on growth and DNA synthesis. Recombinant IL-3 (5-50 u/ml) also showed no significant effect on the incorporation of radioactivity into medium GAG (data not shown). The effect of the addition of glucocorticoid (a soluble cortisol preparation) on GAG production by MC was also studied, in the presence and absence of Con A (at both 4 and 11 day incubation periods). Concentrations of hydrocortisone at 100 pg/ml and 10 pg/ml entirely suppressed the Con A response of MC in terms of both the incorporation of 'H-thymidine into DNA and incorporation of the radioactive precursors into GAG. For concentrations of hydrocortisone that were less than 1.0 (0.01, 0.1, 0.5 was a w/ml 1 there w/ml concentration-dependent decline of incorporation of radioactive precursors into the GAG, in the Con Astimulated cultures (data not shown). The effect
of the R-D-xyloside
(0.05
mM and 0.2mM) on the
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elaboration of PG from MC was also studied, using a continuous labelling protocol (1, 3 and 6 days), in the absence and in the presence of Con A. In the absence of Con A, only relatively small stimulatory effects were observed, the largest of which was at the 0.05 mM concentration after 3 days of incubation and consisted of a 30% increase in 3H and 95% increase in ?SO, incorporation into the CS (data not shown). In the presence of Con A, no additional stimulatory effect was observed but, at a concentration of the B-D-xyloside of 0.2 mM, after 6 days of incubation there was a significant (approximately twothirds) reduction in the stimulation of incorporation of radioactivity from both isotopes into CS. The effect of phorbol myristate acetate on the elaboration of PG was also studied in a manner analogous to the xyloside. At concentrations of 10 rig/ml and 20 rig/ml the phorbol myristate had no effect on PG elaboration at 3 or 6 days of incubation. Discussion
From the data in Figure 1 it appears that the MC PG is elaborated, very largely, by the T-cell population. Serum was required for maximal stimulation of DNA synthesis (Fig. 2) and we had previously shown that serum is also required for maximal stimulation of the elaboration of medium-PG by Con A (Chopra et al, 1991). Also the addition of Con A to MC cultures (incubated in the presence of resulted in increased cell numbers, increased serum) incorporation of 3H-thymidine into cellular DNA and increased incorporation of 3H-glucosamine (and '"SO,) into the CS of the medium GAG (Fig. 3). The addition of IL-2, 100 increased at 20 u/ml or resulted in u/ml, incorporation of 3H-thymidine into the cells, the higher concentration also resulting in increased cell numbers. Others have shown that the elaboration of IL-2 required the expression of the T3/Ti receptor (in a human leukemic T-cell line, Jurkat) and the addition of phorbol myristate acetate acted as a second stimulus for maximal production of IL-2, by increasing intracellular Ca" (Weiss et al., 1987). However, IL-2 had no effect on the incorporation of radioactivity into the CS of the medium GAG (Fig 3), suggesting that cell proliferation alone can not account for the increased amount of labelled PG found in the medium of Con A-stimulated cultures. A similar conclusion was reached with mouse thymocytes, where much smaller stimulations of PG synthesis by phytohaemagglutanin were noted than in the present study, since prostaglandin E, inhibited lymphocyte proliferation but not PG synthesis (Bartold et al., 1989). The
addition
of
supernatants
from
Con A-stimulated
MC
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resulted in enhanced GAG biosynthesis of human synovial fibroblasts (Anastassiades and Wood, 1981) and similar results were obtained when supernatants from activated MC were tested on skin fibroblastic cells in culture (Whiteside et al., 1985). However, the addition of conditioned media from Con A-stimulated or non-stimulated MC failed to stimulate the appearance of radiolabelled PG in the media of MC cultures (see Results, section 3). Also, human IL-l stimulated GAG production by human synovial fibroblasts (Hammerman and Wood, 1984; Yaron et al., 1987), connective tissue activating peptide stimulated hyaluronic acid synthesis by human synovial fibroblasts (Sisson et al., 1980), recombinant gammainterferon, tumour necrosis factor lymphotoxin and stimulated GAG production by human lung fibroblasts (Elias et al., 1988), TGF-I3 stimulated PG synthesis by rat and recombinant calvaria cells (Chopra et al., 1990), human IL-3 stimulated the synthesis of a CS PG by human eosinophils in culture (Rothenberg, et al, 1988). By contrast, PG elaboration was not stimulated in human MC cultures by a partially purified factor from bone containing matrigenin activity (Anastassides et al., 1987), by IL-l, IL-2 and recombinant IL-3, by combinations of IL-l and IL-2, or by TGF-8 (section 3, Results and Fig. the addition of B-D-xyloside to the 3). We found that unstimulated MC cultures resulted in a small increase in incorporation of radioactivity into GAG, while for the stimulated cultures there was a reduction of stimulation in incorporation. In a recent report (Steward et al., 1990) the addition of the nitrophenyl derivative of the A-D-xyloside to isolated T-cells resulted in xylosidestimulation of incorporation of 35so, into associated GAG but in a 50% reduction into PG in phorbol unstimulated cultures. We, further, found myristate acetate had no significant effects on synthesis of the MC PG, at concentrations where it has been reported to be active in other cell systems. The differences in the actions of these agents, on PG elaboration by the MC and suggest that the PG synthesized and by other cell types, secreted by Con A-stimulated MC is under a different control mechanism than the stimulation of PG synthesis of cells responding to peptide growth factors. REFERENCES Anastassiades, T., Irwin, D. and Robertson, W. (1985). The effect of human bone matrix extracts on the biosynthesis of macromolecules by human mononuclear cells in culture. Med. Biol. 63: 123-130. Anastassiades, T. and Wood, A., Elliott, M., Stephens, C and Kisilevsky, R. (1987). Comparison of the stimulation of glycosaminoglycan synthesis in
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cultures of mononuclear cells from blood and of fibroblastic cells. Cell Differ. 21: 37-46. Anastassiades, T.P. and Wood, A. (1981). Effect of soluble products from lectin-stimulated lymphocytes on the growth adhesiveness and glycosaminoglycan synthesis of cultured synovial fibroblastic cells. J. Clin. Invest. 68: 792-802. Anastassiades, T.P. and Wood, A. (1983). Lectin-dependent and lectin-independent activities of human mononuclear cells that modulate the growth of human synovial cells. J. Rheumatology 10: 539-549. Bartold,
P.M.,
Haynes,
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B (1989).
Effect of mitogen and lymphokine stimulation on proteoglycan synthesis by lymphocytes. J. Cellular Physiol. 140: 82-90. Chopra,
R.K., (1991).
Griffith,
E.,
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D.,
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T.
Human T-lymphocytes synthesize and secrete a protease resistant proteoglycan in a delayed serumdependent response to Concanavalin A. Cell Biol. Intern. Rep. 15: 25-35.
Chopra,
R-K., Li, (1990).
Z-W.,
Vickery,
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and
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Newly synthesized proteoglycans secreted by sequentially derived populations of cells from new-born rat calvaria: Effects of TGF-8 and matrigenin activity. Cell Differ. Develop. 32: 4759. T.P.
Elias,
J.A., (1988).
Krol,
R.C.,
Freundlich,
B.,
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Sampson,
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Regulation of human fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor and lymphotoxin. J. Clin. Invest. 81: 325-333. Hammerman, D. and Wood, D.D. (1984). Interleukin-1 synovial cell hyaluronate synthesis. Proc. enhances Sot. Exp. Biol. Med. 177: 205-210. Handwerger, B.S. and Schwartz, R.H. (1974). Separation of murine lymphoid cells using nylon wool columns. Transplantation 18: 545-547. Hronowski, L. and Anastassiades, T. (1979). Quantitation and interaction glycosaminoglycans with alcian blue in dimethyl sulfoxide solutions. Anal. Biochem. 93: 60-72. Hronowski,
Muir, Otten,
L. and Anastassiades, T. (1980). Rates of glycosaminoglycan synthesis and rates of incorporation of radioactive precursors into newly synthesized glycosaminoglycans by confluent rat muscle fibroblasts. J. Biol. Chem. 255: 9210-9217. Proteoglycans as organizers of the H. (1983). The seventeenth CIBA medical intercellular matrix. lecture. Biochem. Sot. Trans. 11: 613-622.
G., Pierce, S.K., F-(1985). Lymphokine
lymphocytes.
Shay,
J.,
Olshan,
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production by cloned In Lymphokines. (Ed. E. Pick)
and
Fitch,
T pp. 13-38,
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New York.
Pomerantz, J.L., Owen, W.F. Jr., Soberman, R.J., Au&en, K.F., and (1988). Characterization of a human
eosinophil proteoglycan, and augumentation of its biosynthesis and size by interleukin 3, interleukin colony stimulating 5, and granulocyte/macrophage factor. J. Biol. Chem. 263: 13901-13908.
Sisson,
J.C.,
Castor,
Connective tissue hylaluronic acid Med. 96: 189-197.
Stevens,
R-L.,
C.W.
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Kamada,
M.&l.
Klavons,
XVIII. activity.
and
J.A.
Serafin,
(1980).
Stimulation of J. Lab. Clin.
W.E.
(1988).
W. and
Galagher,
Structure and function of the family of proteoglycans that reside in the secretory granules of natural killer cells and other effector cells of the immune response. Current Topics Microbial. Immunol. 140: 93108.
Steward, J.T.
W-P., Christmas, S-E., (1990). The synthesis
T-lymphocytes Weiss,
Biochim.
A., Shields, Imboden, J.
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R., (1987).
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T.L., Worrall, J-G., Prince, R-K., Buckingham, and Rodnan, G.P. (1985). Soluble mediators from
Yaron,
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mononuclear cells increase the synthesis of glycosaminoglycans by dermal fibroblasts from normal subjects and progressive systemic sclerosis patients. Arthritis Rheum. 28: 188-197 (1985). I.,
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Human recombinant interleukin-18 glycosaminoglycan production fibroblast cultures. Arthritis Paper received
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stimulates in human synovial Rheum. 30: 424-430.
Revised paper accepted
19.07.91,
international
Reports,
721
Vol. 15, No. 8, 7 99 1
DIFFERFNTIAL EFFECTS OF CONCANAVALIN A, SERUM CYTOKINES ON PROTEOGLYCAN ELABORATION AND DNA SYNTHESIS MONONUCLEAR CELLS FROM HUMAN PERIPHERAL BLOOD Tassos
Anastassiades*, Anne
Departments University,
of Medicine Kingston,
Ravi Chopra, Wood
Mary
and Biochemistry, Ontario, Canada
*Corresponding
Elliott
AND BY and
Queen's K7L 3N6
Author
ABSTRACT
Human blood derived mononuclear cell (MC) cultures required concanavalin A (Con A) stimulation to synthesize and secrete into the medium high levels of a proteaseresistant proteoglycan (PG) containing predominantly chondroitin sulfate (CS), which was elaborated largely by T-cells in culture. PG and DNA synthesis were studied in MC cultures in the absence and presence of Con A as well as serum and some biologically active polypeptide factors. In the presence of Con A, stimulation of PG synthesis was substantially greater in T-cell enriched cultures than in also B-cell enriched cultures. DNA synthesis was stimulated in the presence of Con A. This stimulation was concentration-dependent, but required the presence of serum for additional responses. DNA and cell proliferation were stimulated by interleukin-2 (IL-2), but PG production was not stimulated by conditioned media, IL-l, IL-2, ILgrowth factor-8 (TGF-8). Our ressults 3, or transforming indicate that the elaboration of PG from T-cells of human MC is independent of the effects of regulatory peptides on cell proliferation and DNA synthesis. INTRODUCTION
PG are anionic glycoconjugates which are synthesized primarily by the differentiated cells of the connective tissues and may serve as organizers of the extracellular matrix (Muir, 1983). However, blood leucocytes, including the polymorphonuclear leucocytes, monocytes, and natural killer cells also elaborate PG that are stored in secretory granules (Stevens et al., 1988). In many chronic inflammatory states, such as rheumatoid arthritis, MC accumulate in the affected connective tissues where they are thought to undergo antigenic stimulation. We had reported that PG synthesis was detectable only at a very low level in MC cultures from human blood, but these cultures could be stimulated by Con A to secrete very high the major anionic levels of a CS PG, which represented glycoconjugate elaborated by these cells (Anastassiades et 0309-I
651 I91 /080721-l
l/$03.00/0
0 1991
Academic
Press Ltd
722
Cell Biology
International
Reports,
Vol. 75, No. 8, 199 7
al., 1985, 1987). This PG is protease-resistant, requires the presence of serum for maximal response and appears in the medium relatively late in the culture period, generally between 3-9 days (Chopra et al., 1991). This late appearance of the MC PG is in contrast to the pattern of elaboration of a number of lymphokines, including IL-2 produced by T cell clones, which achieved maximal levels in the medium by 24-48 h after stimulation (Otten et al., 1985), and fibroblast growth suppressive activity from Con A-stimulated human lymphocytes which was detected at significant levels by 17 h of culture (Anastassiades and Wood, 1983). We have undertaken a systematic study of conditions affecting PG elaboration by human MC from the blood and, in this paper, we report on the effects of the addition of IL-l, IL-2 on PG and DNA synthesis in this system, as well as the effects of factors known to stimulate PG synthesis of connective tissue cells, such as TGF-8 and "Matrigenin" activity (Anastassiades et al., 1987). MATERIALS
AND METHODS
Isolation and separation of MC: MC (or l~whole~~ MC) were isolated from the peripheral blood of healthy human volunteers by a Ficoll-Hypaque gradient procedure (Anastassiades and Wood, 1981, 1983) and separated into a T-cell enriched population and a B-cell enriched population on a nylon wool column, as previously described (Handwerger and Schwartz, 1974; Chopra et al., 1991). The individually, or separated populations were cultured llreconstitutedVV in the proportions of B and T cells MC were also present in the "Whole" MC population. separated on the basis of their adherance to plastic into adherent (Ad) and non-adherent (NAd) populations. MC were suspended in appropriate medium, incubated in plastic were removed, dishes for 2h or 24 h, the NAd cells centrifuged, suspended in the appropriate incubation medium and seeded in 2 ml medium/well. Cultures referred to as *VrecombinedV' had the resuspended cells added back to the Ad cells. 1.
2. Preparation of conditioned media: NAd, Ad, recombined or unseparated MC were incubated in serum-free or serumcontaining medium (10% fetal bovine serum, FBS) for 1 to incubation, the supernatants were removed, 6 days. After the cells spun down, the supernatants (conditioned media) were filtered (Nalgene syringe filters, 0.2 /.J) and stored at 4°C. 3.
Metabolic
labelling
*VWholelV MC, separated MC were incubated in
and
treatments
MC populations, 35 mm' plastic
of
cell
cultures:
or "reconstituted" wells in CMRL 1969
Cell Biology
International
Reports,
Vol. 15, No. 8, 1997
723
medium containing 10% FBS and the radioactive precursors of PG (1.25 /Xi/ml 'H-glucosamine, 30-60 Ci/mmole and 2.0 /Xi/ml "S-sulfate, lo-100 mCi/mmole; DuPont-NEN) added at the start of incubation period (continuous labelling protocol). For the pulse labelling protocol 2.5 pCi/ml 'Hglucosamine and 4 pCi/ml "S-sulfate were added 18 h prior to termination of incubation. Incubations were performed in the absence or presence of 12.5 pg/ml Con A, or conditioned media, or substances listed below, added at the begining of the incubation period at the concentrations indicated under Results: IL-l and IL-2 (Intermedico Co.); TGF-B (R and D systems): phorbol 12myristate 13 acetate and methylumbelliferyl-B-D-xyloside (Sigma Chemical Co.): Hydrocortisone sodium succinate (Upjohn Co.). 4. Counting of Ad and NAd MC cell populations: At the completion of incubations, the NAd cell pellets obtained as described above, were suspended in Hematall isotonic diluent (Fisher Scientific) and counted (Coulter Counter Model 3F). The Ad cells at the bottom of the dishes were incubated with 1.5 ml trypsin (0.25% - Gibco) at 37OC until most cells were detached, the suspension was transferred to a tube containing 2 ml medium (10% FBS) and the procedure was repeated, ensuring that no cells remained in the dish as determined by microscopy. 6. into Assay of radioactive incorporation glycosaminoglycans (GAG): After incubation radioactive GAG were isolated from the media and cells following papain digestion, precipitation by cetyl pyridinium chloride, and separation by cellulose acetate electrophoresis as previously described (Hronowski and Anastassiades, 1979, 1980) and the radioactivity incorporated in to the isolated GAG was determined. 7. Incorporation of C3H]-thymidine: MC were suspended in CMRL 1969 medium containing 10% FBS, or in the serum-free medium and seeded into 96-well plates. Cells were treated with varying concentrations of Con A and incubated for 1 to 9 days, followed by pulse labelling with 0.5 @Y/well of [methyl-'HI-thymidine (20 Ci/mmol, DuPont-NEN) for 18 hrs before termination of the culture, the cells were semi-automatic harvester the collected by a and incorporated radioactivity determined as previously described (Anastassiades and Wood, 1983; Anastassiades et al., 1987). RESULTS by MC populations 1. PG synthesis cells: Both T-cell and B-cell
enriched enriched
in T- and Bpopulations
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isolated by the nylon wool column technique and cultured separately, as well as "Whole" MC which not stimulated with Con A (NS), produced ;ery littlewe;: that could be detected in the medium of the cultures (Fig. 1). However, when Con A was added there were large increases in the accumulation of radiolabelled PG in the medium of cultures of 8'wholeVV, T-cell-enriched and V1reconstituted'V populations, while the B-cell-enriched population showed only a modest increase in the PG accumulation. 2.
Effects
of
serum
and
Con
A on
DNA
synthesis
by
MC:
Initial experiments indicated that Ad populations elaborated very little GAG, while NAd populations incorporated as much, or more, radiocativity from 3Hglucosamine and ?3-sulfate into medium CS as the lUreconstitutedl' population. Further, polypeptide "mediators", such as IL-l, which were presumably released from the adherent MC during the early part of the culture period, did not play an important role in the elaboration of PG appearing in the medium in this system (unpublished results). This notion as well as the relationship of the stimulation of DNA synthesis and cell replication in Con *-8 ‘0
Figure 1. Radiolabelled GAG produced by B-Cell-enriched T-Cell-enriched (T), "ReconstitutedVV (B+T) and (B), l'Wholel' (W) MC populations in culture. All cultures contained 2~10~ cells per dish and were incubated for 7 days in continuous culture in medium containing 10% FBS and the radioactive precursors of PG. Replicate (at least 4) culture plates were either not stimulated (NS) or
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725
stimulated with 12.5 pg/ml Con A (Con A). Only 3H counts incorporated into the isolated and separated GAG are shown for clarity as the "S counts followed a similar pattern. The bars indicate standard errors from the mean.
6.
2
4 TIME
6 IN CULTURE
8 ( DAYS
IO 1
8.
2
4 TIME
6 IN CULTURE
8 ( DAYS
IO 1
2. The relation of Con A concentration to jHthymidine incorporation by MC. Con A was added at the indicated concentrations (as pg/ml culture medium) at the begining of the culture period and the cultures were incubated either in 10% FBS-containing medium (A) or in serum-free conditions (B). -_----------------------------------------------------to the accumulation of medium A-stimulated MC cultures, were further PG (measured as radiolabelled GAG) evaluated. DNA synthesis was studied in the MC system, using a standard pulse labelling technique with 'Hthymidine, under both serum-containing and serum-free culture conditions (Fig. 2). For cultures incubated in 2 A) increasing medium containing 10% FBS (Fig. concentrations of Con A led to increased incorporation of generally similar to that 'H-thymidine, in a pattern previously observed for GAG synthesis (Chopra et al., The optimal Con A concentration required to 1991). stimulate incorporation of 'H-thymidine was less for serumfree media (Fig. 2B) than for serum-containing media. 'H-
Figure
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Figure 3. Effects of biologically active peptides on cell numbers, DNA synthesis and GAG synthesis. Ad, NAd cell numbers (A), incorporation of 'H-thymidine into cellular DNA (B) and 'H incorporation from 3H-glucosamine into chodroitin sulfate, CS, and hyaluronic acid, HA (C) were assessed after 7 days of incubation of the MC cultures (pulse labelling protocols). At the beginning of the incubation period Con A, or, a peptide factor was added to the cultures and the additions (or controls) are indicated under the bar graphs in panel C and apply also to A and B. Designations and concentrations are: Con A, 12.5 pg/ml Con A; NS, non-stimulated (controls); 11,(l), IL-l at 0.5 u/ml; at 1.5 u/ml; 11,(l), IL-2 at 20 u/ml; 11,(h), IL(h) r IL-l IL-2 at 100 u/ml; 11,+,(l), IL-1 at 0.5 u/ml and IL-2 at 20 u/ml; IL-1 at 1.5 u/ml and IL-2 at 100 u /ml; 11,+,(h), TGF-I3 at 2 rig/ml; TGF(h), TGF-A at 5 rig/ml. 'J='(l), --------------------------------------------------------thymidine responses to Con A were well developed by 4 days in cultures that contained serum, but were diminished and detectable only at 4 days (at 1.25 I.cg/ml of Con A) if serum was ommitted from the culture (Fig. 2A and 2B).
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3. Comparison substances on
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of effects GAG elaboration
of
some biologically
active
As mentioned in the Introduction, newly synthesized GAG appears in the medium relatively late in the culture period, after Con A stimulation (Chopra et al., 1991). In order to examine this observation further cross-feeding experiments with "conditioned" media were initially carried out. However, the addition of supernatants from Con-A stimulated or non-stimulated Ad, NAd or ~~Whole~~MC cultures to non-stimulated Ad, NAd or "whole" MC cultures failed to stimulate the appearance of radiolabelled GAG in the media of these cultures. Also, there was no additional stimulation of GAG elaboration by Con A when conditioned media (from Ad, NAd or "whole" cultures, both stimulated and non-stimulated with Con A) were used to supplement NAd populations. and
DNA
synthesis:
In order to study the relationship of GAG elaboration to DNA synthesis selected biologically active peptides, that would be expected to be elaborated by MC cultures, were then added, either individually or in combinations, to the culture system (Fig. 3). The addition of the higher concentration of IL-2 (100 u/ml), with or without IL-l, resulted in large increases in the total numbers of cells, primarily the Ad population (Fig. 3 A), and both the high and low concentrations of IL-2 stimulated incorporation of 'H-thymidine into DNA (Fig. 3 B). TGF-A (at concentrations shown to stimulate growth and PG synthesis of fibroblastic cultures, see Discussion) had no effect on growth, DNA synthesis or GAG synthesis in this system. Neither the addition of IL-l nor IL-2 had any significant effect on incorporation of 'H or '?S into the isolated GAG (Fig. 3 C), in spite of the stimulatory effects of these factors on growth and DNA synthesis. Recombinant IL-3 (5-50 u/ml) also showed no significant effect on the incorporation of radioactivity into medium GAG (data not shown). The effect of the addition of glucocorticoid (a soluble cortisol preparation) on GAG production by MC was also studied, in the presence and absence of Con A (at both 4 and 11 day incubation periods). Concentrations of hydrocortisone at 100 pg/ml and 10 pg/ml entirely suppressed the Con A response of MC in terms of both the incorporation of 'H-thymidine into DNA and incorporation of the radioactive precursors into GAG. For concentrations of hydrocortisone that were less than 1.0 (0.01, 0.1, 0.5 was a w/ml 1 there w/ml concentration-dependent decline of incorporation of radioactive precursors into the GAG, in the Con Astimulated cultures (data not shown). The effect
of the R-D-xyloside
(0.05
mM and 0.2mM) on the
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elaboration of PG from MC was also studied, using a continuous labelling protocol (1, 3 and 6 days), in the absence and in the presence of Con A. In the absence of Con A, only relatively small stimulatory effects were observed, the largest of which was at the 0.05 mM concentration after 3 days of incubation and consisted of a 30% increase in 3H and 95% increase in ?SO, incorporation into the CS (data not shown). In the presence of Con A, no additional stimulatory effect was observed but, at a concentration of the B-D-xyloside of 0.2 mM, after 6 days of incubation there was a significant (approximately twothirds) reduction in the stimulation of incorporation of radioactivity from both isotopes into CS. The effect of phorbol myristate acetate on the elaboration of PG was also studied in a manner analogous to the xyloside. At concentrations of 10 rig/ml and 20 rig/ml the phorbol myristate had no effect on PG elaboration at 3 or 6 days of incubation. Discussion
From the data in Figure 1 it appears that the MC PG is elaborated, very largely, by the T-cell population. Serum was required for maximal stimulation of DNA synthesis (Fig. 2) and we had previously shown that serum is also required for maximal stimulation of the elaboration of medium-PG by Con A (Chopra et al, 1991). Also the addition of Con A to MC cultures (incubated in the presence of resulted in increased cell numbers, increased serum) incorporation of 3H-thymidine into cellular DNA and increased incorporation of 3H-glucosamine (and '"SO,) into the CS of the medium GAG (Fig. 3). The addition of IL-2, 100 increased at 20 u/ml or resulted in u/ml, incorporation of 3H-thymidine into the cells, the higher concentration also resulting in increased cell numbers. Others have shown that the elaboration of IL-2 required the expression of the T3/Ti receptor (in a human leukemic T-cell line, Jurkat) and the addition of phorbol myristate acetate acted as a second stimulus for maximal production of IL-2, by increasing intracellular Ca" (Weiss et al., 1987). However, IL-2 had no effect on the incorporation of radioactivity into the CS of the medium GAG (Fig 3), suggesting that cell proliferation alone can not account for the increased amount of labelled PG found in the medium of Con A-stimulated cultures. A similar conclusion was reached with mouse thymocytes, where much smaller stimulations of PG synthesis by phytohaemagglutanin were noted than in the present study, since prostaglandin E, inhibited lymphocyte proliferation but not PG synthesis (Bartold et al., 1989). The
addition
of
supernatants
from
Con A-stimulated
MC
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resulted in enhanced GAG biosynthesis of human synovial fibroblasts (Anastassiades and Wood, 1981) and similar results were obtained when supernatants from activated MC were tested on skin fibroblastic cells in culture (Whiteside et al., 1985). However, the addition of conditioned media from Con A-stimulated or non-stimulated MC failed to stimulate the appearance of radiolabelled PG in the media of MC cultures (see Results, section 3). Also, human IL-l stimulated GAG production by human synovial fibroblasts (Hammerman and Wood, 1984; Yaron et al., 1987), connective tissue activating peptide stimulated hyaluronic acid synthesis by human synovial fibroblasts (Sisson et al., 1980), recombinant gammainterferon, tumour necrosis factor lymphotoxin and stimulated GAG production by human lung fibroblasts (Elias et al., 1988), TGF-I3 stimulated PG synthesis by rat and recombinant calvaria cells (Chopra et al., 1990), human IL-3 stimulated the synthesis of a CS PG by human eosinophils in culture (Rothenberg, et al, 1988). By contrast, PG elaboration was not stimulated in human MC cultures by a partially purified factor from bone containing matrigenin activity (Anastassides et al., 1987), by IL-l, IL-2 and recombinant IL-3, by combinations of IL-l and IL-2, or by TGF-8 (section 3, Results and Fig. the addition of B-D-xyloside to the 3). We found that unstimulated MC cultures resulted in a small increase in incorporation of radioactivity into GAG, while for the stimulated cultures there was a reduction of stimulation in incorporation. In a recent report (Steward et al., 1990) the addition of the nitrophenyl derivative of the A-D-xyloside to isolated T-cells resulted in xylosidestimulation of incorporation of 35so, into associated GAG but in a 50% reduction into PG in phorbol unstimulated cultures. We, further, found myristate acetate had no significant effects on synthesis of the MC PG, at concentrations where it has been reported to be active in other cell systems. The differences in the actions of these agents, on PG elaboration by the MC and suggest that the PG synthesized and by other cell types, secreted by Con A-stimulated MC is under a different control mechanism than the stimulation of PG synthesis of cells responding to peptide growth factors. REFERENCES Anastassiades, T., Irwin, D. and Robertson, W. (1985). The effect of human bone matrix extracts on the biosynthesis of macromolecules by human mononuclear cells in culture. Med. Biol. 63: 123-130. Anastassiades, T. and Wood, A., Elliott, M., Stephens, C and Kisilevsky, R. (1987). Comparison of the stimulation of glycosaminoglycan synthesis in
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Regulation of human fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor and lymphotoxin. J. Clin. Invest. 81: 325-333. Hammerman, D. and Wood, D.D. (1984). Interleukin-1 synovial cell hyaluronate synthesis. Proc. enhances Sot. Exp. Biol. Med. 177: 205-210. Handwerger, B.S. and Schwartz, R.H. (1974). Separation of murine lymphoid cells using nylon wool columns. Transplantation 18: 545-547. Hronowski, L. and Anastassiades, T. (1979). Quantitation and interaction glycosaminoglycans with alcian blue in dimethyl sulfoxide solutions. Anal. Biochem. 93: 60-72. Hronowski,
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