Production of interleukin-1β-like factor with synovial cell growth promoting activity from adult T-cell leukemia cells

Journal of Autoimmunity (1991)

4,223-236

Production of Interleukin-lp-like Factor with Synovial Cell Growth Promoting Activity from Adult T-cell Leukemia Cells

Nobuyuki Miyasaka,* Megumu Higaki,* Kazuto Sate,? Junko Hashimoto,t Atsuo Taniguchi,-f Hitoshi Kohsaka,f Kazuhiko Yamamoto,@ Kanji Shichikawal) and Kusuki Nishiokat *Department Medical

of Virology and Immunology,

and Dental

of Rheumatology, Medicine,

University,

Tokyo;

Tokyo Women’s Medical

Tokyo Medical

and Ph_vsical Therapy,

Medical

tDivision

College,

and Dental University,

Research

Institute,

of Clinical Immunology, Tokyo;

Tokyo Institute

$First Department

Tokyo; $Department

Tokyo University, Tokyo; )IShichikawa Center, Hisai, Mie,Japan

of

of Medicine

Arthritis

Research

(Received 1S May 1990 and accepted 83141~ 1990)

We observed a case of adult T-cell leukemia (ATL) with proliferative synovitis. Culture supernatants from ATL cells (ATL-SN) obtained from the peripheral blood constitutively produced an interleukin-1 (IL-l)-like factor in vitro, as shown by the growth inhibition factor (GIF) assay using the A375 melanoma cell line and the lymphocyte activating factor (LAF) assay using C3H/HeJ thymocytes. Neutralization studies indicated that polyclonal antibodies against IL-18 blocked most (80%) of the activity in ATL-SN. In addition, increased amounts of IL-lg mRNA were found in the ATL cells by dot blot analysis. Sephacryl S-200 chromatography showed that the molecular weight of this factor was approximately 17.5 kDa, and Western blot analysis revealed that this factor reacted with polyclonal anti-IL-lg antibody under the reduced condition. The isoelectric point was 7.5. Furthermore, ATL-SN showed significant activity in promoting the growth of synovial cells in parallel with IL-1 activity. These data suggest that the constitutive production of this IL-lb-like factor might be responsible for proliferative synovitis in this case.

Correspondence Research Institute,

to: Nobuyuki Miyasaka, MD, Department of Virology and Immunology, Medical Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo, Japan. 223

0896-841

l/91/020223+

14 $03.00/O

0 1991 Academic

Press Limited

224 N. Miyasalcaet al.

Figure 1. Marked synovial proliferation in bilateral wrist joints seen in this ATL patient.

Introduction

Adult T-cell leukemia (ATL) is a unique type of leukemia seen mainly in southwestern Japan [l], and human T-cell leukemia virus type I (HTLV-I), a type-C retrovirus, is believed to be a causative agent [2]. HTLV-1 can induce transformation or immortality of human T-cells, which are known to produce multiple cytokines, such as y-interferon ($-IFN), migration inhibitory factor (MIF), macrophage activating factor (MAF), and colony stimulating factor (CSF) [3]. Interleukin-2 (IL-2), interleukin-3 (IL-3) and B-cell growth factors (BCGF) were also reported to be elaborated by HTLV-1 -transformed human T cells [4]. We observed a case of chronic ATL presenting with proliferative synovitis [5]. The patient showed prominent bilateral synovial proliferation in the wrist and elbow joints (Figure l), and histological examination of synovial tissues revealed marked proliferation of synovial cells with leukemic cell infiltration. The majority of the increased leukocytes in the peripheral blood (24,2OO/cm) were medium-sized lymphocytes with large nuclei and some indentation and basophilic cytoplasms (socalled cower-like or ATL cells). We therefore speculated that these ATL cells might produce cytokines capable of inducing synovial cell proliferation. We found that a large amount of interleukin-1 p-like factor with synovial cell growth-promoting activity was constitutively produced by the ATL cells. Materials and methods Subject

The patient was a 79-year-old Japanese woman whose clinical profiles and laboratory data have been described elsewhere [5]. The diagnosis of ATL was established on the

IL-16-like factor twoduction from ATL

225

basis of the following criteria: (1) a majority of the peripheral lymphocytes had convoluted nuclei; (2) these cells expressed CD2 and CD4 antigens, and (3) the patient had antibody to HTLV- 1 in the serum. Preparation of peripheral blood lymphocytes

Peripheral blood mononuclear cells (PBMC) were separated by Ficoll gradient centrifugation as previously reported [6] and resuspended in RPM1 1640 (Gibco autologous serum, Lab., Grand Island, NY) containing 10 “/< heat-inactivated 100 U/ml of penicillin, 100 yg/ml of streptomycin, and 10m~ of hepes (Sigma Chemical Co., St. Louis, MO). These cells proliferated vigorously in response to phytohemagglutinin (1 pg/ml, Difco, Detroit, MI), concanavalin A (10 pg/ml, Sigma) and IL-2 (0.5 ng/ml, Takeda Pharmaceutical Co., Osaka, Japan) (data not shown). Giemsa staining of a blood smear showed that over 98% of the cells were medium-sized lymphoid cells having large nuclei with indentations. Electron microscopy of the cells revealed uniform morphology with prominent indentations in the nuclei. Two-colour analysis with Epics profile (Coulter Corp., Hialeah, FLA) demonstrated that these cells expressed CD2, CD3 and CD4 antigens in addition to HLA-DR antigens and CD25 (IL-2 receptors); they also lacked CDlO, CD14, CD 16, CD20 and CD45R on their surfaces. Contamination by monocytes was minimal ( < lo,) as assessed by esterase staining and cytofluorometry (CD 1lb+ cells < 19,). Various numbers of PBMC were incubated with or without stimulants for 24 to 72 h in 24-well culture plates (Sumitomo, Japan), and culture supernatants (designated as ATL-SN) were harvested at various intervals and assayed for cytokine activity as described below. Assay for IL-l activity

IL- 1 activity was measured by a growth inhibition assay using the melanoma cell line A375 [7, 81. Briefly, A375 cells were plated into 96-well flat bottom culture plates (1 x lo4 cells/well) in Eagle’s MEM supplemented with 10% fetal calf serum (FCS) with various dilutions of test samples or recombinant IL-l p (kindly provided by Dr Yoshikatsu Hirai of Otsuka Pharmaceutical Co. Ltd). After 4 days’ incubation at 37”C, 0.059, neutral red was added to each well, and when incorporated in viable cells was extracted with ethanol after 2 h of culture. The optical density of each well was measured at 540 nm by a multiscan photometer. One unit of growth inhibitory factor (GIF) per ml represented the reciprocal of the dilution of samples, causing 50”, cytostasis or cytolysis after 4 days of culture. Five hundred pg/ml of recombinant IL-lp resulted in 100°/’ cytostasis. The specificity of the assay was further confirmed by using recombinant human tumour necrosis factor a (TNFa), IL-2 and IL-6. With this assay, 2 to lOOOpg/ml of IL-1 was detectable. IL-l activity was also confirmed by a thymocyte proliferation assay using concanavalin A-stimulated thymocytes of C3H/He J mice (LAF assay) [9]. Assay for IL-2 activity IL-2 activity was assayed by using an IL-2-dependent

[lOI.

mouse cytotoxic T-cell line

226

N. Miyasaka et al. Assay for IL-6 activity

IL-6 activity was determined by using an Epstein-Barr line, SKW6-Cl-4 cells as described previously [ 111.

Assay for synovial cellgrowth

virus-transformed

B-cell

promoting activity

The synovial cells were obtained by arthroscopic synovectomy from the knee joint of a patient with classical rheumatoid arthritis. The synovial tissue was minced, washed extensively with phosphate-buffered saline (PBS), and treated with PBS plus 0.25% trypsin for 40 min at 37°C. The cells were washed three times with PBS and finally suspended in HAM F-12 medium (GIBCO) supplemented with 10% FCS, 5 x lop5 M 2-mercaptoethanol (2-ME), 100 U/ml of penicillin and 100 pg/ml of streptomycin. The cells were incubated in culture flasks until they became confluent, then treated with 0.05% trypsin for 5 min at room temperature. Morphologically, these cells were a mixture of fibroblast-like cells, macrophage-like cells, and dendritic cells. After they were adjusted to 2 x lo4 cells/ml with 10% FCS-RPM1 1640, 100 l.rlof the cell suspension was plated in 96-well culture plates with various concentrations of test samples or recombinant IL- 1p for 72 h, and neutral red uptake was measured by the method described above. In preliminary experiments, using L cells asindicator cells instead of synovial cells, this assay was shown to have a significant correlation between cell number and neutral-red uptake into the cells after a 5-day culture period (data not shown).

Neutralization

of IL-l

activity

by anti-IL-l

antibodies

ATL-SN with prominent IL-l activity were mixed with various anti-IL-l-antibodies to determine whether their ability to inhibit the A375 melanoma cell line was neutralized by these antibodies. Anti-human recombinant IL-l antibodies were kindly provided by Dr Hirai. OCT303 and OCT204 were polyclonal anti-human recombinant IL-la and anti-human recombinant IL-lp rabbit antibodies, respectively [ 121. These antibodies were highly specific and did not react with IL-2, insulin, or epidermal growth factor.

Dot blot analysis of IL-l

mRNA

Fresh ATL cells were lysed in isothiocyanate [ 131and homogenized. Total RNA was recovered after centrifugation through cesium chloride. RNA extracted from mononuclear cells obtained from buffy coat layers of the peripheral blood was used as a control. The concentration of RNA was determined by absorbance at 260 nm. Equal amounts of RNA were subsequently transferred onto nitrocellulose filters after being serially diluted, and filters were prehybridized in a solution containing 50”/; formamide, 5 x SSC (sodium chloride, sodium citrate buffer), 20 mM phosphate buffer (pH 6.5), 0.1 mg/ml denatured DNA from salmon sperm in 1 x Denhardt’s solution at 42°C and hybridized overnight with 32P-labelled cDNA probe (pcDGIF-207 for IL-la and pcD-GIF-16 for IL-lp, respectively) [14] at 42°C. Blots

IL-lblike factor xwoductionfrom ATL

227

were washed twice using high stringency conditions (0.1 x SSC, 0.1% sodium dodecyl sulfate SDS at 65°C for 1 h) before being exposed to X-ray film. Gel filtration

One million PBMC were cultured in 1y0 FCS-RPM1 1640 at 37°C for 24 h without any stimulant. After incubation, ATL-SN were collected by centrifugation at 400 g for 10 min and concentrated lo-fold by an Amicon Diaflo membrane (YMlO, MW cutoff 10,000). Then 2.0 ml of concentrated ATL-SN was applied to a 1.6 x 90 cm column of Sephacryl S-200 (Pharmacia, Sweden) equilibrated with PBS. The flow rate was adjusted to 15.0 ml/h and 2 ml fractions were collected. The column was calibrated with bovine serum albumin (MW 66,000), carbonic anhydrase (MW 29,000), cytochrome C (MW 12,400), and aprotinin (MW 6,500). The fractions were passed through 0.45~pm filters and assayed for cytokine activities. Western blot analysis

Ten-fold concentrated ATL-SN was mixed with 2 x SDS buffer containing 0.125 M tris base, 4.6% SDS, 5% 2-ME, and 20% glycerol, and boiled for 5 min. Twentyfive ~1of samples were run on a 12.5% (weight/volume) polyacrylamide gel containing SDS. After electrophoretic transfer to nitrocellulose, strips were treated with polyclonal rabbit anti-human IL- la antibody or anti-human IL- 18 antibody (1:750 final dilution, respectively) or rabbit IgG, followed by treatment with goat antirabbit IgG (Tago Inc., Burlingame, CA). Antibody binding was visualized using peroxidase-conjugated swine anti-goat IgG (Tago) followed by addition of the substrate, 3’,3’-diaminobenzidine. Chromatofocusing

Three hundred ~1of the concentrated samples were applied on a PBE94 (Pharmacia) column (2 x 15 cm) equilibrated with 25 mM Tris-acetate (pH8.2) and eluted with 100 ml of the eluent buffer containing Polybuffer 96: Polybuffer 74 (3:7) adjusted to pH 5.0 with acetate at a flow rate of 30 ml/h. Aliquots of 2 ml were collected by monitoring 0D280 and the pH. After extensive dialysis against PBS (-), each fraction was tested for IL-l activity in the assay as described. Results Fresh A TL cells produce a growth inhibitory factor (GIF) A375

for melanoma cell line

One million PBMC were incubated for various culture periods (24 to 96 h) in the presence or absence of PHA. SN were collected by centrifugation and assayed for cytokine activities. No IL-2 activity was detected when an IL-2-dependent CTL cell line was used, and only negligible amounts of IL-6 activity (less than 0.5 U/ml) were found in these ATL-SN using SKW-Cl-4 cell line. However, ATL-SN cultured for 24 h in the absence of PHA strongly inhibited the growth of the A375 melanoma cell

228 N. Miyasaka et al.

E .o

80

80

5 3 e UJ 40

:, ‘S P AZ .E c 5 e 0

z

%

z 2

80

x

40

x 20

20

8 Concentration

Figure ATL-SN

80

32

125

500

of human IL-l/3 (w/ml)

4 Reciprocal

2. A375 melanoma cells were incubated with various dilutions for 4 days as described in Methods. Results of a representative

16

64

256

dilution of sample

of (a) recombinant IL-lp, experiment are shown.

or (b)

line (48 U/ml) (Figure 2), which is equivalent to the activity exerted by 500 pg/ml of recombinant IL-lb. As A375 cells react specifically with IL-I but not with other cytokines, including tumour necrosis factor a (TNFa), ATL-SN are suggested to possess an IL-l-like factor. The production of the IL-l -like factor was observed even with 1 y0 autologous serum, although its amount was approximately 50% lower. This IL-l-like activity was consistently found in SN cultured up to 96 h, although there was a slight decrease in activity with time. In addition, stimulation of ATL cells with PHA did not alter the production of IL-l-like factor but induced inhibitory factor(s) in the GIF assay (data not shown). For further analysis, we therefore decided to use ATL-SN cultured for 24 h in the absence of PHA.

A TL-SN

also possessed LAF activity

ATL-SN supported the proliferation of Con A-stimulated thymocytes of C3H/HeJ mice in a dose-dependent fashion (Table 1). The LAF activity reached its maximum on day 1 of culture and gradually tapered with length of culture in parallel with GIF activity. ATL-SN lacked IL-2 activity, so it is conceivable that the LAF activity was attributed to IL-l.

IL-l-like

activity

can be neutralized

by polyclonal antibody against IL-la IL-la

but not

We next tested whether the IL-l-like activity could be inhibited by polyclonal antibodies against IL-la or IL-l p. Ten thousand A375 cells were incubated with 25 l.tlof ATL-SN and either anti-IL-la or anti-IL-l p polyclonal antibody. Rabbit IgG with an equivalent protein concentration was used as a negative control. Approximately 80% of the GIF activity was blocked by anti-IL-l /3polyclonal antibody but neither

IL-lfi-like factor production from ATL

229

Table 1. ATL-SN induced the proliferation of Con A-stimulated thymocytes of C3He/J mice

Sample None Recombinant IL-l p (1 ng/ml) ATL-SN (1:8) ATL-SN(1:16) ATL-SN (1:32) ATL-SN ( 1:64)

Proliferation of thymocytes (cpm) 600’ 7805 9469 6671 5816 3072

‘Mean value of triplicate cultures. Standard deviationswere less than lOobof mean values.

Table 2. Neutralization of IL-l-like activity by antiIL-1 antibodies in GIF assay Sample ATL-SN ATL-SN+anti-IL-la antibody’ ATL-SN+anti-IL-lb antibody ATL-SN + rabbit IgG

GIF activity (U/ml) 50 47 10 48

IFinal concentrationof antibodieswas adjustedto 12.5 pg/ml.

anti-IL-la nor rabbit IgG affected GIF activity (Table 2). The combination of anti-IL-la and anti-IL-l p antibodies did not augment inhibition by anti-IL-lp antibody. Moreover, addition of either anti-IL- la antibody or anti-IL- 1b antibody without ATL-SN had no affect on the growth of A375 cells. This suggests that IL-l-like activity might be derived from IL-ll3.

Absence of IL-l -like factor production from an ATL cell line establishedfrom PBMC of the same patient As fresh ATL cells expressed IL-2 receptors (CD25 antigen), as shown by cytofluorometry, and vigorously proliferated in response to recombinant IL-2 (Takeda Pharmaceutical Co., Ohsaka, Japan), we tried to establish long-term ATL cell lines from PBMC of the same patient by culturing this PBMC with 20% PHAstimulated culture supernatants of PBMC obtained from multiple healthy subjects. One cell line with the same phenotype as fresh ATL cells was established after a month of culture and was completely dependent on IL-2 for proliferation. However, the cell line did not demonstrate IL-l-like factor in any of the culture conditions tested.

230

N. Miyasaka et al.

Figure 3. Expression of IL-l mRNA in ATL cells. RNA was extracted from either a buffy coat layer of the peripheral blood from a healthy donor or ATL cells. RNA was subsequently transferred to nitrocellulose filters after serial dilution and hybridized with a 32P-labelled cDNA probe for either IL- la (Figure 2a) or IL-l p (Figure 2b).

Fresh A TL cells contain a large amount of IL-lb mRNA

Total RNA was extracted from ATL cells, and equal amounts of RNA were subsequently transferred onto nitrocellulose filters to be hybridized with 32P-labelled cDNA probes for either IL-la or IL-ll3. As shown in Figure 3, whereas a large amount of IL-lfl mRNA was present in fresh ATL cells, there was only a small amount of IL- 1a mRNA. Control RNA obtained from buffy coat did not hybridize with either of the cDNA probes. Fresh A TL cells producedfactor (s) with syn&ial cell growth-promoting activity

This ATL patient showed prominent synovial proliferation, as shown in Figure 1, and histopathological examination of the synovial tissues revealed marked proliferation of synovial cells adjacent to massive infiltration of ATL cells. We therefore hypothesized that ATL cells could induce synovial proliferation by producing soluble factors with synovial cell growth-promoting activity. Synovial cells were obtained from rheumatoid synovial tissue (as described in the methods section) and cultured for approximately 2 weeks. When over 4 x lo3 cells/well were cultured in 10% FCS-RPMI, they spontaneously proliferated and their growth was not dependent on exogenous growth factor. However, when fewer than 4 x lo3 cells/well were cultured, they did not proliferate significantly without stimulation in a 4-day culture. We therefore decided to use this culture condition to test the ability of ATL-SN to induce synovial cell proliferation. Cultured synovial cells were plated (2 x lo3 cells/ well) and incubated with various concentrations of ATL-SN or recombinant IL- 1p or recombinant IL-6 for 96 h at 37°C. ATL-SN strongly promoted the growth of synovial cells in a dose-dependent fashion (Figure 4), and this capacity was more potent than that of 1 ng/ml of recombinant IL-ll3. Moreover, this capacity was

IL-l@like factor production from ATL

8

128

64

16

Reciprocal

dilution

231

of sample

Figure 4. Rheumatoid synovial cells (2 x lo4 cells/well) were incubated with various concentrations of ATL-SN (0), or recombinant IL-l (O), or recombinant IL-6 (A) for 96 h at 37°C as described in Methods. Anti-IL-l p antibody abrogated synovial cell growth promoting activity of ATL-SN (+).

2’.

8

i0

12

14

16

18

20

22

24

26

28

30

32

Figure 5. A lo-fold-concentrated ATL-SN was chromatographed on Sephacryl S-200. The fractions collected were tested for IL-l activity using A375 melanoma cells (0) or synovial cell growth promoting activity (0). Anti-IL-10 antibody completely abolished theiractivity(A).

blocked by addition of polyclonal anti-IL-l/3 antibody to the culture. In contrast, recombinant IL-6 did not induce synovial cell proliferation in this assay system. Biochemical

characterization

of ATL-SN

A lo-fold-concentrated ATL-SN cultured in ly; FCS-RPM1 for 24 h was chromatographed on Sephacryl S-200. The fractions collected were tested for both

232

N. MiyasaJca et al.

Figure 6. Western blot analysis of IL-l activity in ATL-SN. onto 12.5% SDS-polyacrylamide gels under the reduced nitrocellulose, the sample was treated with rabbit antiserum nant IL-@, or with rabbit IgG, followed by treatment with visualized using peroxidase-conjugated swine anti-goat IgG diaminobenzidine.

A 1O-fold-concentrated ATL-SN was run condition. After electrophoretic transfer to to human recombinant IL-la or to recombigoat anti-rabbit IgG. Antibody binding was followed by addition of the substrate 3’,3’-

3.0 60 c

5

7.0

z D

6

6.0%

x 20

5.0 s

10

I5 Fraction

20

25

30

3s

number

Figure 7. A IO-fold-concentrated ATL-SN was applied on a PBE94 column. dialysed against PBS and tested for IL-l activity using A375 melanoma cells.

Fractions

collected

were

IL- 1 activity and synovial cell growth-promoting activity. The peak of IL- 1 activity was eluted from the column at the position corresponding to a molecular weight of approximately 17.5 kDa (Figure 5). This fraction also had the maximal ability to promote the growth of rheumatoid synovial cells. Moreover, synovial cell growthpromoting activity was abolished by anti-IL-l p antibody. When recombinant IL-l p

IL-l&like factor production

from ATL

233

was chromatographed on this column, the peak IL-l activity was eluted from the same position as ATL-SN (data not shown). A IO-fold-concentrated ATL-SN was further run onto 12.596 SDS-polyacrylamide gels under the reduced condition. When anti-IL- 1p antibody, but not anti-ILla antibody or rabbit IgG, was applied to a nitrocellulose filter after transfer, a clear band was seen at approximately 17.5 kDa and faint bands at between 30 and 40 kDa (Figure 6). Subsequently, the isoelectric point value of IL-l activity of ATL-SN was determined by the chromatofocusing technique. GIF activity was eluted as a single peak at approximately pH 7.5, suggesting that the IL-l-like molecule in ATL-SN corresponds to IL-l p (Figure 7). Discussion Our results indicated that fresh ATL cells produced an IL-l P-like factor capable of inducing vigorous proliferation of synovial cells. ATL cells strongly inhibited the growth of A375 melanoma cells in GIF assay and induced DNA synthesis of Con Astimulated thymocytes of C3H/HeJ mice in LAF assay. Most of the GIF activity of ATL-SN was neutralized by the polyclonal antibody against IL-1s but not IL-la. Moreover, Western blot analysis demonstrated a band reactive with anti-IL-l p antibody at approximately 17.5 kDa under the reduced condition. The isoelectric point of the IL-l-like factor also corresponded to that of IL-lb. Furthermore, increased mRNA of IL-l@ was demonstrated by dot blot analysis using a cDNA probe for IL- 1fl. These data suggest that IL- 1-like activity in ATL-SN might be attributable to IL-1p. It is unlikely that contamination by macrophages was responsible for production of the IL-IP-like factor in ATL-SN. Macrophages represented less than 1O,, of peripheral blood lymphocytes as assessed by esterase staining and cytoflourometry (CD 11b ’ < 1Ob). In addition, removal of phagocytic cells by ingestion of silica particles did not affect the production of the IL-lP-like factor in ATL-SN (data not shown). In this respect, Wano et al. [ 151 reported IL- 1J3gene expression in freshly isolated primary tumour cells, but not in long-term cell lines from ATL patients, using Sl nuclease protection assays; however, they did not mention the clinical features of their patients. This finding is in complete accordance with our results. Production of IL-la, but not IL-l p, by ATL cell lines was also reported by Yamashita and his colleagues [16], who also indicated that peripheral blood lymphocytes from ATL patients produced a cytokine with bone resorbing activity corresponding to IL-la [17]. Increased IL-l mRNA expression was detected not only in HTLV-l-transformed T-celI lines but also in Epstein-Barr virus-transformed Bcell lines, suggesting that viral infection itself might activate IL- 1 genes [ 181. Noma er al. [ 191 recently described increased amounts of IL-l mRNA detected in nine out of 16 ATL cell lines examined. However, IL-l production for T cells may be encountered not only in pathological situations but also under physiological conditions. Tartakovsky et al. [20] demonstrated that murine T-cell clones produce IL-l after stimulation by accessory cells. These data suggest that IL-l gene expression can occur even in T cells following proper activation signals, although the subspecies of IL-l produced may differ in each case.

234 N. Miyasaka et al.

We have already shown enhanced IL-l production from biopsied rheumatoid synovium [lo] and cloned synovial cells from rheumatoid joints [21]. Increased IL-l production from rheumatoid synovium correlated well with the proliferation of synovial cells, which strongly expressed HLA-DR antigen [lo]. IL-l is known to promote proliferation of fibroblasts [22], and the results of this study also indicate that IL-l, but not IL-6, induces proliferation of rheumatoid synovial cells. Of note, production of an IL-lP-like factor from ATL cells gradually tapered, in parallel with a decrement in synovial cell growth-promoting activity, with the cessation of synovial proliferation (data not shown). This might support the notion that the production of an IL-l P-like factor contributed to the synovial proliferation seen in this case. Multiple growth factors have been described for fibroblasts. Spontaneous production of fibroblast-activating factor(s) (FAF) by synovial inflammatory cells has been shown by Wahl et al. [23, 241. FAF were detected by gel filtration in two fractions with molecular weights of 40 kDa and 15 kDa. These authors speculated that the former peak might be derived from T cells and the latter from monocytes, although they did not mention whether these factors are analogous to IL-l. In this regard, Salahuddin et al. [3] have revealed that HTLV-l-infected T-cell lines produce a cytokine with FAF activity, although they also indicated that their LAF activities were low. Other cytokines, such as fibroblast growth factor (FGF), insulinlike growth factor (IGF), epidermal growth factor (EGF), transforming growth factor j3 (TGFP), and platelet-derived growth factor (PDGF), promote fibroblast growth [25]. TNFa also has fibroblast growth-enhancing activity [26]. In this aspect, Butler et al. [27] demonstrated that IL-la, IL-lfi, TNFa, TNFP, and interferon-y induced DNA synthesis of synovial fibroblasts obtained from rheumatoid arthritis patients. They further reported that PDGF and basic FGF are the potent stimulators of synovial fibroblast proliferation [28]. In contrast, Lafyatis et al. [29] showed that PDGF stimulates whereas TGFP inhibits synovial cell growth. Among these growth factors for fibroblasts, PDGF needs to be further discussed. It is released not only by platelets but also by activated macrophages in response to injury and/or inflammation [30]. Raines and Ross [31] clearly demonstrated that the mitogenic activity of IL-l for fibroblasts is mediated by PDGF-AA, one of the isoforms of PDGF. It is therefore possible that the IL-l P-like factor produced by ATL cells in our study exerts its effect via PDGF. The possibility that an IL-l P-like factor in ATL-SN stimulates synovial cells to produce PDGF is currently being investigated in our laboratory. Finally, our collaborators recently disclosed a unique clinical feature of chronic inflammatory arthropathy associated with HTLV-1 infection [32]. All the patients were HTLV-1 carriers with proliferative synovitis. Atypical lymphocytes, compatible with ATL-like cells, were observed in both fluids and synovial tissues. It is possible that HTLV-1 infected T cells could stimulate the production of synovial cell growth factor, leading to arthropathy, as shown in this case. Further study is being undertaken. Acknowledgements

This work was partly supported by a grant-in-aid from the Ministry of Health and Welfare, Japan. We are grateful to Dr Yoshikatsu Hirai at Otsuka Pharmaceutical Company for supplying polyclonal antibodies against IL-l and cDNA probes for

IL-Q-like factor production from ATL

235

IL- 1, and to Dr Toshio Hirano and Dr Tadamitsu Kishimoto of the Institute for Molecular and Cellular Biology, Osaka University, for providing IL-6-dependent cell lines and anti-IL-6 antibodies. We also thank Mr Kazumasa Ikeda of Japan Scientific Instrument Company for monoclonal antibodies and Miss Hiroko Inoue of Mitsubishi BCL Co. Ltd for her technical assistance.

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