modulation by recombinant interleukin 1 of synthesis of types i and

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 262, No. 34, Issue of December 5, pp. 16724-16729,1987 Printed in U.S.A.

Modulation by Recombinant Interleukin 1 of Synthesis of Types I and I11 Collagens and Associated Procollagen mRNA Levels in Cultured Human Cells*

(Received for publication, October 31, 1986)

Mary B. Goldring and Stephen M. Krane From the Department of Medicine, Harvard Medical School and the Medical Services (Arthritis Unit), Massachusetts General Hospital, Boston, Massachusetts 021 14

Interleukin 1 (IL-1), a monocyte product, exerts a range of biological effects on nonimmune cells such as fibroblasts and chondrocytes including stimulation of synthesis and release of prostaglandin Ez (PGE2) and collagenase. We have previously shown that crude mononuclear cell-conditioned medium, which contains IL- 1, also stimulates synthesis of types I and I11 collagens by human synovial and dermal fibroblasts and chondrocytes when the formation of PGE2, which inhibits collagen synthesis, is blocked by indomethacin. To determine whether IL-1 is responsible for the effects observed using crude monocyte-conditioned medium patterns of collagen synthesis in the three types of human cells in response to recombinant preparations of IL- 1 were compared. Preincubation of chondrocytes or synovial fibroblasts with either murine (m)IL-la or human (h)IL-l@ alone decreased synthesis of type I collagen and fibronectin. In contrast, when endogenous IL-1-stimulated PGE, synthesis was blocked by indomethacin, an enhancing effect of IL-1 on synthesis of these matrix proteins was unmasked. The synthesis of type I11 collagen was enhanced by IL-1 to a greater extent than that of type I collagen in the presence of indomethacin. In human foreskin fibroblasts, which produced low levels of PGE, even in the presence of IL-1, synthesis of types I and I11 collagens was increased by IL-1 either in the absence or presence of indomethacin. These cells were more responsive to the hIL-l@ preparation than to the mIL-la (half-maximal stimulation of PGEz production was observed at -2.5- 5 p~ hIL-I@ and at -2.5 nM mIL-la). Levels of al(I), a2(I), and a1 (111) procollagen mRNAs measured by cytoplasmic dot hybridization paralleled the levels of collagens synthesized under the various experimental conditions. IL- 1, therefore, is one product of monocytes capable of modulating collagen synthesis by these human mesenchymal cells probably by altering collagen gene expression. These studies suggest that both positive (IL-1) and negative (PGEz) signals may control collagen synthesis at the transcriptional level resulting in modulation of matrix turnover in cartilage, synovium, and skin.

* This work was supported by United States Public Health Service Grants AM-03564 and AM-03490 and Research Service Award AM- 07258. This is Publication 1032 of the Robert W. Lovett Memorial Unit for the Study of Diseases Causing Deformities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Interactions of inflammatory cells and connective tissue target cells play a major role in the destruction of the extra- cellular matrix in inflammatory diseases such as rheumatoid arthritis (1, 2). There is evidence for repair of connective tissue associated with the inflammatory process, and this repair process is probably also regulated by interactions be- tween inflammatory cells and connective tissue cells (3).

One product of inflammatory cells, interleukin 1 (IL-l),’ has been detected in joint effusions from humans with rheumatoid arthritis and osteoarthritis (4) and is released by rheumatoid synovial tissue in vitro (5). IL-1, a product pre- dominantly of monocyte/macrophages, stimulates the synthesis and release of prostaglandin Ez (PGE2) and neutral pro- teases including collagenase and plasminogen activator by human synovial cells and chondrocytes (6-12). Previous work from this laboratory showed that crude mononuclear cell- conditioned medium, which contains IL-1, increases synthesis of types I and I11 collagens and fibronectin by synovial cells when the formation of PGE2, which decreases collagen synthesis (13), is inhibited by indomethacin (3). Such crude or partially purified preparations from cultured peripheral blood cells contain other inflammatory cell products including the T lymphocyte product interferon (IFN-7) which inhibits collagen synthesis and suppresses procollagen mRNA levels in synovial and dermal fibroblasts and chondrocytes (14-18).

In order to determine whether IL-1 could be responsible, at least in part, for these effects of monocyte-conditioned medium on matrix synthesis it is necessary to utilize recombinant preparations of this cytokine. The cDNAs for the two gene products of IL-1 have been cloned and expressed (19,20). In the present studies primary cultures of human articular and costal chondrocytes, as well as synovial and foreskin fibroblasts, were, therefore, used as target cells in a model system to examine the effects of recombinant IL-la and -p on collagen synthesis and procollagen mRNA levels. We show that IL-la and -6 are two components of monocyte-conditioned medium which can account for some of the previously observed effects on these human cells.

EXPERIMENTAL PROCEDURES~

RESULTS

To determine if IL-1 could be responsible for any of the previously observed effects of monocyte-conditioned medium

The abbreviations used are: IL-1, interleukin 1, mIL-loc, murine interleukin la ; hIL-18, human interleukin 18; PGE,, prostaglandin E*; IFN-y, interferon-y; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis; TCA, trichloroacetic acid; 8-ME, 8- mercaptoethanol.

* Portions of this paper (including “Experimental Procedures” and

16724

Collagen Synthesis by Interleukin 1 -treated Human Cells 16725

Day 21

" X "

0 0 + + 0 0 mILIOC 0 + 0 + 0 + Indo 0 0 0 0 + + MonoCM

Day 27 I'

0 0 + + o 0 o + o + o + o o o o + +

FIG. 1. Effects of mIL-la, monocyte-conditioned medium (mnoCM), and indomethacin (Indo) on pepsin-resistant [3H] proline-labeled collagens synthesized by articular chondrocytes. Chondrocytes (from a 79-year-old female) in 35-mm diameter dishes on (A) day 20 or ( B ) day 26 of primary culture were preincubated for 24 h alone or with indomethacin M), mIL-la (100 pM), mIL-la plus indomethacin, monocyte-conditioned medium, or monocyte-conditioned medium plus indomethacin. [3H]Proline was then added for a further 24 h, and pepsinized medium (50 pl of original medium) was analyzed by SDS-PAGE (5% acrylamide) without and with delayed reduction. Only unreduced gels are shown. Delayed reduction showed that the unreduced bands migrating with the y-collagen standard were consistent with type I11 collagen. Films were exposed to gels for 13 days.

on collagen synthesis, human articular chondrocytes in mono- layer culture were used to examine the effects of recombinant IL-1 on synthesis of types I and I11 collagens and production of PGE, and collagenase. In Fig. 1 is shown a representative experiment in which the responses to arbitrary concentrations of mIL-la and monocyte-conditioned medium were compared in articular chondrocytes at two different times in late primary culture. Articular human chondrocytes were found previously to synthesize little or no type I1 collagen (18). In these articular chondrocytes, whereas either indomethacin or a submaximal concentration (100 pM) of mIL-la alone slightly increased collagen synthesis above control levels, addition of indomethacin unmasked a stimulatory effect of mIL-la on synthesis of types I and I11 collagens. Addition of monocyte- conditioned medium alone resulted in a marked inhibition of type I collagen synthesis which, compared with the indomethacin control, was only partially reversed by indomethacin. These results were typical of those previously obtained using monocyte-conditioned medium and chondrocytes isolated from 20 different specimens of articular cartilage each exam-

Tables 1-111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request DOC- ument No. 86M-3767, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

ined at 1-4 different times during primary culture or after subculture. A marked suppression of type I collagen synthesis by monocyte-conditioned medium was observed in >90% of the experiments, while addition of indomethacin with monocyte-conditioned medium resulted in at least a partial reversal of this suppression, and in some cases (-25%) a stimulatory effect was unmasked (data not shown). The suppression of collagen synthesis which was reversed by indomethacin could be accounted for in part by stimulation of PGEz synthesis by IL-1 or monocyte-conditioned medium (Table I, Miniprint). The levels of collagenase in the medium were also increased by mIL-la or monocyte-conditioned medium either in the absence or presence of indomethacin (Table I). None of these differences could be accounted for by changes in cell number or DNA content which did not change significantly under the experimental conditions used. Chondrocytes isolated from four different specimens of adult articular cartilage were examined with respect to their responsiveness to IL-1 each after different periods of primary culture and in one case after subculture. Results consistent with those described above and below were obtained in all experiments. When the labeled cell layers were analyzed by SDS-PAGE following pepsinization, the pattern of labeled bands reflected those in the medium.

To further investigate the inhibitory effects of PGEz on collagen synthesis, increasing concentrations of mIL-la were added to a different preparation' of articular chondrocytes from another individual in the absence or presence of indomethacin (Fig. 2). In the absence of indomethacin, mIL-la produced a dose-dependent decrease in collagen synthesis. This suppression was probably accounted for by the high ambient levels of PGE, induced by IL-1 (Table 11, Miniprint), since addition of indomethacin reversed the inhibition by mIL-la alone and unmasked a dose-dependent stimulation. In the experiment shown in Fig. 2, 100 PM mIL-la alone suppressed collagen synthesis compared to the lack of suppression at the same concentration in the experiment described in Fig. 1. This is possibly accounted for by the increased PGE, response to mIL-le in the earlier experiment (27-fold uersus 14-fold). Recombinant hIL-16 also decreased collagen synthesis in these human chondrocytes in the ab-

dl (1)-

ci 2(I) -

w

QI

d U/ml mlkla! ,O 0.5 1.0 5 IO 50,,0 0.5 1.0 5 IO 50,

w/o Indo + Indo FIG. 2. Effects of preincubation with mIL-la in the absence

and presence of indomethacin (Indo) on collagen synthesis by articular chondrocytes. Chondrocytes (female, 65-year-old) at day 21 of primary culture were preincubated for 48 h alone or with recombinant mIL-la at 0.5 units/ml (50 pM), 1.0 unit/ml (100 pM), 5 units/ml (500 PM), 10 units/ml (1.0 nm), or 50 units/ml (5.0 nM) in the absence or presence of indomethacin. Incorporation of [3H] proline and SDS-PAGE of pepsin-resistant collagens was carried out as described in Fig. 1. Only the gel using delayed reduction is shown. Film was exposed to the gel for 4 days.

16726 Collagen Synthesis by Interleukin 1 -treated Human Cells

A Articular Synovial Foreskin

Chondrocytes Fibroblosts Fibroblosts Unreduced Reduced Unreduced Reduced Unreduced Reduced

- FN

0 + O + O + O + - I n d o - O + O + O + O + - I n d o - O + O + O t O t

B Articular Synovial Foreskin

Chondrocytes Fibroblasts Fibroblasts

. -r

Lmb

0 -k 0 + "Indo- 0 + 0 + -1ndo - 0 + 0 + 0 0 + + "hIL-1/3 - 0 0 + + -hIL-1/3 - 0 0 + +

FIG. 3. Effects of hIL-1D and indomethacin (Indo) on ['HI proline-labeled procollagens, collagens, and fibronectin synthesized by human articular chondrocytes and synovial and foreskin fibroblasts. Articular chondrocytes (day 21 of primary culture, see Fig. 2), synovial fibroblasts (11 days after second subculture), and foreskin fibroblasts (4 days after 4th subculture) were preincubated for 48 h alone (0) or with (+) M indomethacin, hIL-la (0.5 nM), or hIL-1fl plus indomethacin. A, total medium proteins (25 p1 of original medium) were analyzed by SDS-PAGE without or with reduction with 0.1% P-mercaptoethanol. The migra- tion positions of fibronectin ( F N ) under nonreducing conditions and reducing conditions are indicated. B, pepsin-resistant collagens (50 pl of original medium) were analyzed by SDS-PAGE with delayed reduction. Films were exposed to the gels for 4 days.

sence of indomethacin but increased collagen synthesis in the presence of indomethacin (Fig. 3 and Table 111, Miniprint). The lowest concentration of hIL-lP assayed in these experiments (5 pM) produced maximal stimulation of collagen synthesis in the presence of indomethacin (data not shown), whereas this concentration was approximately only half-maximal with respect to stimulation of PGE, synthesis (Table 11). The response to hIL-lP with respect to collagen synthesis remained maximal at concentrations up to 0.5 nM but fell off slightly at 2.5 nM (data not shown).

Responses of human synovial and foreskin fibroblasts to hIL-lP were then compared to those of articular chondrocytes. Synovial fibroblasts responded to mIL-la and hIL-16 (Fig. 3 and Table 111) in a manner similar to that of articular chondrocytes. hIL-lP alone decreased synthesis of procollagens and fibronectin (Fig. 3A). When ambient levels of endogenous PGE, were decreased in the presence of indomethacin, the stimulatory effect of IL-1 on synthesis of these matrix proteins was unmasked. SDS-PAGE of pepsin-resistant collagens synthesized by these cells demonstrated a marked stimulation of synthesis of both types I and I11 collagens by hIL-16 in the

presence of indomethacin (Fig. 3B and Table 111). In contrast to the results with chondrocytes and synovial cells, in human foreskin fibroblasts collagen and fibronectin synthesis was increased either in the absence or presence of indomethacin (Fig. 3, A and B). As shown in Table 11, these foreskin fibroblasts produced relatively low concentrations of PGEZ even in the presence of IL-1. All of these human cell types were particularly responsive to the human IL-1 preparation; approximately 200-1000-fold higher concentrations of mIL- la compared to hIL-lP were required to increase synthesis of PGE, production to the same extent (Table 11). The half- maximal concentration of hIL-lP with respect to stimulation of PGE, and collagenase production by chondrocytes or synovial fibroblasts was found to be -2.5 PM (data not shown). The effects of mIL-la or hIL-16 on production of collagenase and PGE, by the synovial fibroblasts used in this experiment were not parallel (Table 11).

0 1 1 ( I ) oc2(I) OIl(IU1

Control

Indo

m I L -lo(

mIL-lo(+ Indo h I L -Ip

hIL-I/S+Indo

Control

I n d o

m I L - I d

mIL- Id+ Indo

hIL-l/O+Indo

Control

Indo

rnIL -la mIL-IM+Indo

h I L - I p

hIL-I/B+Indo

FIG. 4. Dot blot hybridization of 32P-labeled al(I), a2(I), and al(II1) procollagen probes to RNA from human articular chondrocytes and synovial and foreskin fibroblasts. Parallel cultures of the cells described in Fig. 3 were incubated for 24 h in 100-mm dishes in Dulbecco's modified Eagle's medium with 10% fetal calf serum (5 ml/dish) alone or with indomethacin (Indo, M), mIL-la (5 nM), or hIL-10 (0.5 nM) with or without indomethacin following which the cells were harvested with 0.25% trypsin and washed and the cytoplasmic RNA was extracted. RNA from cells containing the equivalent of 0.5, 0.25, and 0.125 pg of DNA was applied to nitrocellulose paper in vertical rows 1,2, and 3, respectively. Although the response to mIL-la appears to he greater than to hIL- 16 in some cells, most notably the foreskin fibroblasts, maximal effects to hIL-lB could he observed at concentrations as low as 5 pM (see Table 111) whereas the concentration of mIL-la used was maximal (see Fig. 2 and Table 111). The blot was exposed to film for 16 h with intensifying screen.


In order to determine if the effects of IL-1 and PGE2 were exerted at translational or pretranslational steps in collagen synthesis, steady-state concentrations of procollagen mRNAs were determined. Analysis of the levels of cytoplasmic mRNAs using dot blot hybridization with procollagen DNA probes is shown in Fig. 4 and Table 111. Levels of al(I1) procollagen mRNA in these late primary cultures of articular chondrocytes were too low to determine responses to IL-1. In the articular chondrocytes and synovial fibroblasts, preincubation with indomethacin increased the levels of al(1) and a2(I) procollagen mRNAs, whereas preincubation with mIL- la or hIL-lp alone decreased these levels. When IL-la or -p and indomethacin were added together, however, an increase in type I procollagen mRNAs was observed which was greater than that with indomethacin alone. In the articular chondrocytes, levels of al(1) procollagen mRNA were increased by indomethacin alone or IL-1 in the presence of indomethacin and decreased by IL-1 alone to a greater extent than a2(I) procollagen mRNAs resulting in increased ratios of a l ( I ) / a2(I) procollagen mRNAs (1.1 in control cells versus 1.5 indomethacin-treated and 2.2 in IL-1-treated cells). Similar results were obtained in cultures from two other specimens of articular cartilage with 2.04-3.5-fold increases in al(1) procollagen mRNA in cells treated with IL-1 and indomethacin. In contrast, the levels of al(II1) procollagen mRNA were very low in all cell types examined relative to the type I collagen mRNAs and were unchanged in the presence of indomethacin. In chondrocytes and synovial fibroblasts, there were, therefore, no detectable effects of IL-1 alone on this gene product. Preincubation of these cells with IL-la or -6 in the presence of indomethacin, however, resulted in an increase in al(II1) procollagen mRNA levels which was particularly striking in the chondrocytes and a 2-fold increase in the ratio of al(III)/ al(1) procollagen mRNAs (0.15 in control cells uersus 0.28 in cells treated with IL-1 and indomethacin). The ratios of al(III)/al(I) procollagen mRNAs were also increased in cells treated with IL-1 alone (0.69 in mIL-la-treated and 0.87 in hIL-lp-treated cells) due to the striking decrease of a1(I) procollagen mRNA by IL-1 alone. In the foreskin fibroblasts, a l ( I ) , a2(I) , and al(II1) procollagen mRNA levels were increased by IL-1 either in the absence or presence of indomethacin.

DISCUSSION

The results presented here demonstrate that recombinant IL-1 is capable of inhibiting or stimulating collagen synthesis by cultured human chondrocytes and synovial and foreskin fibroblasts under the appropriate experimental conditions and that these effects are modulated by prostaglandins. Either mIL-la or hIL-1@ alone decreased collagen synthesis by chondrocytes and synovial cells, ascribable to the increased ambient levels of PGE, induced by IL-1. In contrast, a dose- dependent stimulation of collagen synthesis was unmasked when cells were pre-exposed to IL-1 in the presence of indomethacin. Collagen synthesis by foreskin fibroblasts, which produced low levels of PGE2, was increased by IL-1 even in the absence of indomethacin. These results are in agreement with those previously reported (3) and indicate that IL-la and -p are factors present in monocyte-conditioned medium which could be responsible for increasing collagen synthesis in the presence of indomethacin in cultured chondrocytes and synovial fibroblasts.

The two complementary DNAs for human IL-1 have been cloned, sequenced, and expressed as distinct polypeptide

chains which possess IL-1 biological activity measured by the stimulation of proliferation of cloned murine lymphoma cells in the presence of phytohemagglutinin (20, 21). These a and p forms of IL-1 are products of different IL-1 genes. In human blood mononuclear cells, hIL-1p accounts for >95% of the IL-1 produced. In terms of deduced amino acid sequence, the mIL-la (19) shows approximately 62% homology with hIL- la. In our studies, we showed that at least three of the previously reported biological effects of crude monocyte-conditioned medium on human chondrocytes and synovial cells, i.e. stimulation of collagenase and PGE2 production and stimulation of collagen synthesis, are properties of hIL-l@. The latter was observed in some cells only when PGE, synthesis was blocked. The results using mIL-la suggest that human IL- la would have similar effects, although we have not yet tested this possibility. Recent studies have also shown that recombinant preparations of IL-1 stimulate production of PGE2 and collagenase by human synovial cells and fibroblasts (33-36). We found recently that hIL-1p stimulates collagenase synthesis in articular chondrocytes associated with increases in procollagenase mRNA levels (12).

Products of mixed cultures of antigen- or mitogen-stimulated lymphocytes and monocytes have been shown under different experimental conditions to increase (37-40) or decrease (41) collagen synthesis by fibroblasts. A possible expla- nation for these discrepant results was suggested in subse- quent work by Jimenez and co-workers (14, 15) who showed that immune interferon (IFN-r), a product of T lymphocytes, decreases collagen synthesis by fibroblasts. The results of previous work from this laboratory (3) and other laboratories (42) indicated that increased ambient PGE, levels induced by partially purified medium from monocyte-enriched leukocyte cultures could also be partially responsible for this inhibition. In our present studies a stimulation of collagen synthesis by recombinant IL-1 in the presence of indomethacin was con- sistently observed. Purified IL-1 derived from human monocytes has also been shown to stimulate type IV collagen production by epithelial cells (43) and mesangial cells (44). Discrepant results in earlier studies of different investigators (45, 46) could be accounted for by the presence of other cytokines contaminating even highly purified preparations, excessive concentrations of IL-1 employed, or differences in the responses of the particular target cells studied. The inhibition of collagen synthesis in our chondrocyte cultures by the relatively crude preparations of monocyte-conditioned medium not completely reversed by indomethacin could be accounted for by the presence of IFN-7 released by T lymphocytes or other factors released by other types of cells contaminating the monocyte-enriched blood mononuclear cell cultures which were the source of the conditioned medium.

In agreement with previous studies (3) we found that monocyte-conditioned medium or recombinant IL-1 in the absence or presence of indomethacin increased the ratio of type 111 to type I collagen synthesized by chondrocytes. Alterations in the type as well as amount of collagen synthesized was also observed previously in cocultures of pulmonary macrophages with lung fibroblasts in which the ratio of type 111 to type I collagen synthesized was increased (47).

Crude or partially purified monokines as well as purified and recombinant IL-1 stimulate replication of several different cell types including dermal fibroblasts (48), synovial cells (491, and bone cells (50). In some of these cells the stimulation of replication is enhanced by indomethacin. In our chondrocyte cultures, however, cell number and DNA content did not change significantly under the experimental conditions used and did not account for the degree of stimulation of collagen

16728 Collagen Synthesis by Interleukin 1 -treated Human Cells

synthesis. It is recognized, however, that increases in the number of collagen-synthesizing cells is a critical factor in the genesis of fibrotic states (51). Other factors present in Serum such as insulin-like growth factor I (somatomedin C) or platelet-derived growth factor can increase both DNA and collagen synthesis (52). The effects of IL-1 would thus be additive to those of somatomedins or platelet-derived growth factors. Transforming growth factor @ has also been shown to increase collagen and fibronectin synthesis in vitro and fibro- sis in vivo (53).

We have demonstrated here that the increase by IL-1 and decrease by PGEZ of type I collagen synthesis in our human cell cultures are associated with parallel changes in steady- state levels of al(1) and a2(I) procollagen mRNAs. We do not yet know, however, whether these effects are due to changes in the rate of transcription of the procollagen genes or the stability of the mRNAs. Our results also suggest that the levels of al(1) and a2(I) procollagen mRNAs are not regulated coordinately. We showed previously that IFN-y decreased the levels of al(1) procollagen mRNA to a greater extent than a2(I) procollagen mRNAs in rheumatoid synovial fibroblasts (16) and articular chondrocytes (18). In the present studies, levels of al(1) procollagen mRNA were increased by IL-1 in the presence of indomethacin to a greater degree than those of a2(I) procollagen mRNA in articular chondrocytes, although not in synovial or foreskin fibroblasts.

The suppression of collagen synthesis by PGE, in chondrocytes and synovial cells is probably mediated by cAMP as a second messenger. In human synovial cells (49) or chondrocytes (54) pre-exposure to monocyte-conditioned medium or PGE, results in loss of responsiveness, or desensitization, to further stimulation with PGE,, whereas preincubation with monocyte-conditioned medium or IL-1 in the presence of indomethacin results in enhancement of PGE,-stimulated adenylate cyclase activity. The direction of these changes is similar to those of cell proliferation (49) and collagen synthesis (3). Alterations in the cAMP responsiveness to PGE, could be ascribable to changes in number or distribution of cell surface receptors for PGE,. The failure of the small amount of PGE, produced by IL-1-stimulated foreskin fibroblasts to modulate collagen synthesis and procollagen mRNA levels in these cells may be related to differential responsiveness of their receptors compared with synovial fibroblasts and chondrocytes.

Assuming that the changes in the levels of procollagen mRNAs observed are the result of parallel changes in transcription of collagen genes, the mechanisms by which these changes occur must be defined. Human IL-la and IL-lP have been shown to have similar bioactivities in human fibroblasts (55) and act via a specific high affinity receptor present in low concentration (56-58). Presumably IL-1 through its specific receptors must elicit some signal which acts at the level of the collagen gene promoter. IL-1 stimulates synthesis of prostaglandins synergistically with phorbol esters and diacyl- glycerol analogues (59) indicating that these effects may be controlled by one or both limbs of the phosphoinositol lipid pathway (60). The signal resulting from the transduction events and ultimately affecting collagen synthesis could be a protein that interacts with the promoter region of the collagen gene such as has been described for the regulation of proen- kephalin gene expression by cAMP and phorbol ester (61).

In conclusion, we have shown that IL-1 is one factor present in human monocyte-conditioned medium responsible for stimulation of collagen synthesis in human cell cultures. These and previous studies suggest that there is transcrip-

tional control of collagen synthesis which is either positive (IL-1) or negative (PGE, or IFN-y) resulting in modulation of matrix turnover in cartilage, skin, and synovium.

Acknowledgments-We thank James Birkhead for technical assist- ance and Michele Angelo for preparation of the manuscript.

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