MIRICK & G. J. FRIOU Department ofMedicine, California College of ... California College of Medicine, University of California, Irvine, California 92717, USA. 3I2 ...
Clin. exp. Immunol. (1986) 66, 312-319
Increased production of an interleukin 1 (IL-1) inhibitor with fibroblast stimulating activity by mononuclear cells from patients with scleroderma CHRISTY I. SANDBORG*, MONIQUE A. BERMAN, B. S. ANDREWS, G. R. MIRICK & G. J. FRIOU Department of Medicine, California College of Medicine, Irvine,
California (Acceptedfor publication 10 June 1986)
SUMMARY We have previously demonstrated low IL-1 activity produced by peripheral blood mononuclear cells (PBMC) from patients with scleroderma (Sandborg et al., 1985) and the production of a 6-9 K IL-I inhibitor by normal monocytes (Berman et al., 1986). To determine whether this inhibitor accounted for the low IL-1 activity present in scleroderma, the production of IL- I and IL- I inhibitor by PBMC from eight scleroderma patients was studied. Concentrated supernatants from 24 h cultures of unstimulated PBMC were fractionated on Sephacryl S-200 and tested for IL-l and IL-1 inhibitor activity in the standard IL- 1 thymocyte proliferation assay. In seven of eight patients, IL-1 inhibitor production was increased (average 3 3 x) compared to matched controls. IL-1 production was less than controls in six ofeight patients. Partially purified preparations of the 6-9 K mol. wt IL-I inhibitor were inhibitory to IL-I induced thymocyte proliferation but stimulatory to fibroblast proliferation when purified by gel chromatography and chromatofocusing (pI 4-5-5 6). These data suggest that an IL-1 inhibitor with fibroblast stimulating activity is produced in higher amounts by PBMC from patients with scleroderma, and may contribute to the fibroblast proliferation and excessive collagen synthesis which is typical of this disease.
Keywords scleroderma progressive systemic sclerosis interleukin 1 interleukin 1 inhibitor fibroblast stimulating activity
INTRODUCTION The effects of the cytokine IL-1 on the immune and inflammatory responses (Dinarello, 1985) and on the stimulation of fibroblast proliferation (Postlethwaite, Lachman & Kang, 1984) have suggested a role for IL- I in the pathogenesis of scleroderma. Alcocer-Varela, Martinez-Cordero & Alarcon-Segovia (1985) have found elevated levels of IL-1 activity in supernatants of PBMC from scleroderma patients with disease duration less than 5 years and normal levels of IL-1 in patients with long-standing disease. In contrast, we have found lower IL-I activities in PBMC cultures from patients with scleroderma (Sandborg et al., 1985). Our observation that IL-1 activity often increased upon dilution of the supernatant suggested to us the presence of inhibitors of IL-I in the supernatants. Further investigations revealed the presence of a 6-9 K mol. wt factor that inhibited IL-1 activity on thymocyte proliferation (Berman et al., 1986). This inhibitor was produced by Correspondence: Christy I. Sandborg, MD, Department of Medicine, Medical Sciences California College of Medicine, University of California, Irvine, California 92717, USA. 3I2
I, Room C 348.
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normal adherent cells along with IL-I, and had no inhibitory effect on IL-2 dependent cell line proliferation. The present study demonstrates that the 6-9 K IL- 1 inhibitor, while inhibiting thymocyte proliferation, parodoxically results in the stimulation of fibroblast proliferation. To investigate whether this IL-l inhibitor could contribute to the fibroblast proliferation and excessive collagen synthesis typical of scleroderma, IL-1 and IL-1 inhibitor production by mononuclear cells from patients with scleroderma was measured. Functional assays revealed that increased amounts of the IL- I inhibitor and reduced IL-I activity are produced in scleroderma, a pattern which could not be demonstrated in normals or in patients with active juvenile rheumatoid arthritis. METHODS Patient population. Patients in this study with scleroderma were followed at the Scleroderma Clinic at the University of California Irvine Medical Center. Healthy laboratory personnel served as normal controls and patients with active juvenile rheumatoid arthritis provided a control population with another rheumatic disease. Approved written consent was obtained from all patients and controls in the study. Preparation of mononuclear cells. Fresh heparinized whole blood was obtained and mononuclear cells separated by Ficoll-Hypaque (Pharmacia, Piscataway, New Jersey, USA) density centrifugation. After washing three times with Hanks' balanced salt solution (Gibco, Grand Island, New York, USA), the mononuclear cells were resuspended in RPMI 1640 (Gibco), containing 10% fetal calf serum (FCS) (Gibco), 2 mM L-glutamine (Gibco), 5 x 10-5 M 2-mercaptoethanol (2-ME) (Bio Rad, Richmond, California, USA) and 2 ,ug/ml gentamicin (Schering, Kenilworth, New Jersey, USA). Using nonspecific esterase staining (Yam, Li & Crosby, 1971), there was no significant difference in the number of monocytes between patients and controls. Preparation of PBMC culture supernatants. Mononuclear cells were incubated for 24 h at 370C in a humidified 5% CO2 atmosphere at 106 cells/ml. In some experiments, adherent cells were separated from nonadherent cells by adherence to plastic. Nonadherent cells were further purified by passage through Sephadex G- I0 (Pharmacia) (Ly & Mishell, 1974). Supernatants were frozen at - 20OC until further testing. Fractionation of PBMC culture supernatants. Supernatants were concentrated 10-15-fold on Amicon YM-2 membranes (Amicon, Danvers, Massachussetts, USA). In order to fractionate relatively small volumes of supernatant, a sterile miniature Sephacryl S-200 (Pharmacia) column system (4 mm x 270 mm) was used. Sephacryl S-200 gel was autoclaved to sterilize it before use. Fractions (160 pil) were eluted with sterile normal saline. Columns were calibrated with bovine serum albumin (mol. wt 67,000), ovalbumin (mol. wt 43,000), chymotrypsinogen A (mol. wt 25,700) and lysozyme (mol. wt 13,700). Partial pur'ification ofIL-] inhibitor. IL- 1 inhibitory activity was partially purified from human PBMC culture supernatant as previously described (Berman et al., 1986). Inhibition of IL- I induced thymocyte proliferation was found predominately in the 6-9 K mol. wt range. These fractions were pooled, reconcentrated and stored at - 20°C until further use. Chromatofocusing of IL-I inhibitory activity. An anion exchange gel column system eluted by ampholytes to form a pH gradient was used for separating partially purified IL-I inhibitor by its isoelectric point. A sterile miniature column (4 mm x 270 mm) containing Polybuffer Exchanger 94 gel (Pharmacia) was washed with 15 volumes of 0 025 M Tris (Schwarz-Mann, Spring Valley, New York, USA) adjusted to pH 8-5 with 1 M acetic acid. Partially purified IL-1 inhibitor concentrate (500 p1) was loaded onto the column and eluted with a buffer comprising Polybuffer 96 (30% by volume) (Pharmacia) and Polybuffer 74 (70% by volume) (Pharmacia) adjusted to pH 4 5 with 1 M acetic acid. Twenty drop fractions were collected until the pH of the eluant stabilized at pH 4 5. Fractions were adjusted to pH 7 4 by the addition of NaHCO3. Thymocyte proliferation assay. IL-I activity in supernatants was assayed in the thymocyte proliferation assay. Thymic cell suspensions from 4-6 week old Balb/C mice (colony established by the Department of Microbiology, University of California, Irvine, California, USA) were prepared
Christy L Sandborg et al.
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aseptically. Thymocytes were washed and suspended in RPMI 1640 containing 10% FCS, 5 x 10m 2-ME, 2 MM~L-glutamine, and 2y~g/ml gentamicin. Thymocytes (106 cells in 100 PI) were placed in each well of a 96-well microtitre plate (Cell Wells, 25860 Corning, New York, USA) together with 50 pI of 1: 100 dilution of bacto phytohaemagglutinin P (PHA) (Difco Laboratories, Detroit, Michigan, USA) and 25 p1 of the sample column fraction to be tested. To test for inhibitory activity, 1 unit (corresponding to 50% maximal thymocyte proliferation) of partially purified IL-lI prepared as previously described (Berman et al., 1986) was added. Cultures were incubated for 72 h in a 380C humidified CO2 atmosphere, pulsed with 1 pCi 3H-thymidine (3 H-TdR) (6-7 Ci/mmol, New England Nuclear, Boston, Massachussetts, USA) during the last 24 h of culture, and harvested by an automatic cell harvester onto glass fibre filters for counting in a liquid scintillation counter. Fibroblast proliferation assay. Confluent human fetal foreskin fibroblast cultures from passages 4 to 12 were detached from culture flasks with trypsin-EDTA (Gibco), washed and resuspended in RPMI 1640 with 1% FCS, 2 MM L-glutamine, 5 x 10 -5m 2-ME, and 2 pg/mI gentamicin at 105 fibroblasts/ml. Fibroblast suspension, 100 pI, were dispensed into each well of a 96-well flatbottomed microtitre plate and allowed to attach for 1-2 h. Aliquots (25 pI) of samples to be tested were then added, cultures were incubated in a humidified 5% CO2 atmosphere for 72 h, and pulsed with 1 pCi 3H-TdR in the last 24 h of culture. For harvesting, the supernatant from each well was aspirated and 0- I ml trypsin-EDTA was added to each well. Plates were incubated for 10 min until all cells detached, harvested with an automatic cell harvester onto glass fibre filters, and counted. Statistical methods. Standard deviations of the mean ct/mmn from triplicate cultures were obtained. Wilcoxon signed rank test for comparing paired samples of patients and controls was used where indicated.
30
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10 20 3040 5060 10 20 3040 5060 Fraction no.
Fig. 1. Comparison of IL-I and IL-I inhibitory activity from fractionated (Sephacryl S-200) culture supernatant of PBMC from (a) control and (b) scleroderma patient No. 6. Each fraction (20 pl) was tested in the thymnocyte proliferation assay in the presence of 1 U IL- I (177,868 ± 10,432 ct/mmn, mean ± 1 s.d.). Background thymnocyte proliferation without added IL- I was 30,827 ± 1,245 ct/mmn. The hatched area represents total IL-I inhibitory activity (1000 ct/mmn/mm2) and the stippled area represents total IL-i activity (1000 ct/mmn/mm2).
IL-1 inhibitor with fibroblast stimulating activity
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Table 1. Comparison of IL-I inhibitory activity between scleroderma patients and controls IL- I inhibitory activity Patient/control pairs (Ratio: patient/control)* 02 23 13 32 22 52 76 12
2 3 4 5
6 7 8
P value < 0 05 calculated by the Wilcoxon signed rank test for analysis of paired samples. * Ratio of total IL-1 inhibitory activity calculated from supernatant fractionations from patients with scleroderma compared to matched controls.
RESULTS
Patient population. Eight patients were studied: all fulfilled the American Rheumatism Association criteria for the diagnosis of scleroderma. No patients were taking corticosteroids at the time of the study and only one patient (No. 2) was taking d-penicillamine. The remainder were variably on nonsteroidal anti-inflammatory agents (four patients), slow channel calcium blocking agents (three patients) and cimetidine (one patient). Separation of IL-] activity and IL-] inhibitory activity. To separate IL- I activity from inhibitory activity, supernatants from 24 h cultures of PBMC from patients and controls were concentrated 10 to 15-fold, and fractionated on sterile Sephacryl S-200 columns. Each fraction was tested in the thymocyte proliferation assay for IL-I and inhibitory activity. In Fig. 1, which is representative of seven experiments, the scleroderma patient's PBMC produced significantly greater amounts of Table 2. Comparison of the ratio of IL-1 activity to IL-1 inhibitory activity in patients with scleroderma and controls
Patient/controlpairs 1 2 3 4 5 6 7 8
Patients inhibitor*
IL-l/IL-i
19 40 06 25 1.9 04 06 17
Controls inhibitor*
IL-i/IL-i
06 5.1 1.9 194 46 46 46 30
P value < 0-05 calculated by the Wilcoxon signed rank test for analysis of paired samples. * Ratio of areas of total IL-I activity to total IL-I inhibitory activity from supernatant fractionation profiles.
Christy L Sandborg et al.
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inhibitory activity than the normal control. To quantify the relative amounts of IL-l and IL-1 inhibitor, the areas of the total IL- I activity and IL- I inhibitory activity for each patient and control were calculated from these fractionation profiles as shown in Fig. 1. The area in mm2 (1000 ct/min/ mm) above the 1 U IL-I standard was defined as total IL-I activity and the area in mm2 below 1 U IL-1 was defined as total IL-1 inhibitory activity. Each patient was compared to the matched control assayed on the same day. The ratio of total inhibitory activity from each patient sample compared to its control was calculated and is shown in Table 1. All but one patient (No. 1) had significantly more total IL- I inhibitory activity than their control (P < 0 05), including patient No. 2 on d-penicillamine. The ratio of IL-1 activity to IL-1 inhibitory activity was calculated for each subject as this reflects the net IL-1 activity determined in the thymocyte assay. In Table 2, seven of eight scleroderma patients had lower IL-i/IL-I inhibitor ratios than their paired control (P < 0 05). This corresponds to the low IL-1 activity found in crude unfractionated supernatants of scleroderma patients as previously described (Sandborg et al., 1985). IL-1 activity was lower than normal in six of eight patients but these results did not reach statistical significance (P > 0 05). One patient with early scleroderma (patient No. 7) had normal IL-I activity, and the only patient in the group who was taking d-penicillamine at the time of the study (patient No. 2) had twice normal IL-I activity. To determine if these changes were specific for scleroderma or represented a general response in a chronic inflammatory illness, nine patients with active juvenile rheumatoid arthritis were also studied. There was no significant difference in IL-1 or IL-1 inhibitor production between these patients and normal controls. The effect of IL-I inhibitor on fibroblast proliferation. The effect of IL-I inhibitor on fibroblast proliferation was studied for its potential significance in inducing an increase in collagen synthesis characteristic of scleroderma fibroblasts. The IL-1 inhibitor was isolated by Sephacryl S-200 gel chromatography with mol. wt 6-9 K and tested for its ability to inhibit IL-I induced fetal foreskin fibroblast proliferation. Table 3 shows the results of three experiments using IL- I inhibitor purified from three different healthy individuals. These experiments indicate that the IL-I inhibitor did not inhibit IL-I induced fibroblast proliferation, but paradoxically stimulated fibroblast proliferation. To determine whether the fibroblast stimulatory activity was due to contaminating IL-1, to other fibroblast proliferative factors or to the IL-1 inhibitor itself, further studies were done. Adherent and nonadherent cells from healthy volunteers were cultured separately for 24 h and the supernatants concentrated and fractionated on small sterile Sephacryl S-200 columns. Figure 2 is a representative experiment obtained from one of three healthy individuals. Fibroblast stimulatory activity from adherent cell supernatants eluted in two peaks, a broad peak with mol. wt 17-60 K (A) and a second peak with mol. wt 6-9 K (B) coeluting with a peak of IL-I inhibitory activity as determined by inhibition of thymocyte proliferation (C). Nonadherent cell supernatants contained Table 3. Effect of IL-I inhibitor on fibroblast proliferation assessed
by uptake of 3H-thymidine
Incorporation of 3H-thymidine (ct/min) Addition to culture of Experiment I Experiment 2 Experiment 3 Medium IL-I* IL- I + IL- I inhibitort IL-1 inhibitort *
1 unit
3166 + 1691: 7345+352 8433 + 371 6532 + 621
1165 +721 7221 +830 ND 6275 +524
623 + 72 2799+237 4333 +42 2925 +230
IL-I.
t Amount of IL-I inhibitor required to inhibit 1 unit of IL-1induced thymocyte proliferation by 50% 1 Mean value+ 1 s.d.
IL-I inhibitor with fibroblast stimulating activity 9 9.
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Fig. 2. The effect on thymocyte and fibroblast proliferation of supernatants from different mononuclear cell populations. PBMC were separated into adherent and nonadherent populations by plastic adherence and by passage through Sephadex G- 10 before culturing for 24 h. The supernatants were collected and fractionated on Sephacryl S-200. Each fraction was tested in the fibroblast proliferation assay (0 0) with background 3Hthymidine incorporation of 1165 + 125 ct/min (mean + 1 s.d.), and in the thymocyte proliferation assay (0 - - 0) in the presence of 1 U of purified IL-1. 3H-thymidine incorporation in the presence of 1 U of IL-I was 96,485 + 8,012 ct/min in the thymocyte proliferation assay. (A) and (B) mark the peaks of fibroblast stimulation in the 17-60 K mol. wt and the 6-9 K mol. wt range, respectively. C marks the nadir of inhibition of thymocyte proliferation induced by the 6-9 K mol. wt IL- 1 inhibitor. No fibroblast stimulatory factors were present in the cell-free culture medium with 10% FCS incubated for 24 h, concentrated, fractionated on Sephacryl S-200 chromatography and tested in the fibroblast proliferation assay (data not shown).
neither fibroblast proliferative activity nor IL- I inhibitory activity in the 6-9 K mol. wt range. Only adherent cells produced both the fibroblast stimulatory activity and the IL-I inhibitory activity in the same molecular weight range. Further purification of the IL-I inhibitor by chromatofocusing demonstrated that the pI of both the fibroblast stimulatory activity and the IL- I inhibitor were the same (pI 4 5-5 6). Thus the two activities could not be separated by either chromatofocusing or mol. wt. This suggests that both functional activities may reside in the same molecule.
DISCUSSION This study reveals three major findings; first, that PBMC from scleroderma patients produce significantly more IL-I inhibitor than normal controls; second, that the IL-1 inhibitor does not inhibit fibroblast proliferation but rather is mitogenic for fibroblasts; and thirdly, this pattern of ILI and IL-I inhibitor appears to be specific for scleroderma, as we have not demonstrated this in patients with active juvenile rheumatoid arthritis or AIDS (unpublished observations). Our previous study of low IL- I activity in crude supernatants from patients with scleroderma (Sandborg et al., 1985) led to the subsequent description of a 6-9 K mol. wt IL-1 inhibitor produced by adherent cells (Berman et al., 1986). The present study confirms that PBMC from scleroderma patients produce greater amounts of this IL- 1 inhibitor than normals which accounts for the low net IL-I activity in crude supernatants determined by the standard IL-I assay.
3I8 Christy I. Sandborg et al. The increase in IL-I inhibitor production without a concomitant increase in IL-i production appears to be specific for scleroderma as it is not found in juvenile rheumatoid arthritis or AIDS. Increased inhibitor production could result from increased numbers of a certain subpopulation(s) of monocytes responsible for releasing this inhibitor or from changes in the activation/maturational state of the monocytes. Several studies have proposed that monocytes are 'preactivated' in scleroderma (Alcocer-Varela et al., 1985; Andrews et al., 1986), while others have reported the presence of a suppressor monocyte subpopulation in scleroderma (Segond et al., 1982; Lockshin et al., 1983). It is possible that the IL-I inhibitor we have defined may mediate some of the suppressive effects ascribed to the monocyte subsets. The IL-I inhibitor specifically inhibits IL-I induced thymocyte proliferation. It has no effect on IL-I dependent T cell proliferation or peripheral blood T cell mitogenic responses (Berman et al., 1986). This study shows that this IL-I inhibitor has no inhibitory effect on IL-I induced fibroblast proliferation, but rather stimulates fibroblast proliferation. Purification to homogeneity will be necessary to prove definitively that both activities (inhibition of IL-1 induced thymocyte proliferation and stimulation of fibroblast proliferation) reside within the same molecule, although the presence of similar mol. wt and isoelectric points suggests the molecules may be identical. Other investigators have demonstrated that human monocytes produce fibroblast stimulating factors of various molecular weights (Postlethwaite et al., 1984; Estes et al., 1984; Dohlman et al., 1985). The 17-60 K mol. wt fibroblast stimulating activities probably contain a combination of IL- I and other fibroblast stimulating factors (Postlethwaite et al., 1984; Estes, Pledger & Gillespie, 1984; Dohlman, Payan & Goetzl, 1984). The 6-9 K mol. wt fibroblast proliferative factor we have described may be identical to the 10 K mol. wt factor produced by PHA-stimulated human monocytes (pl 4-5-2) (Dohlman et al., 1984). A similar mol. wt factor with a higher pI (5 4-5 6) was produced by the human promonocytic cell line U937 (Dohlman et al., 1985). Further investigation of this factor produced by a single cell source may be helpful in determining the relationships of these fibroblast proliferative factors as well as their effects on IL-i induced thymocyte proliferation. This IL-i inhibitor/fibroblast stimulator and IL- 1 inhibitors described by others (Dinarello, Rosenwasser & Wolff, 1981; Liao et al., 1985; Arend, Joslin & Massoni, 1985) may be important in regulating the multiple in-vivo activities of IL-I. Potential effects of medications taken by the scleroderma patients on the production of IL-I and IL-1 inhibitors were considered. The one patient treated with d-penicillamine at the time of the study had increased levels of IL-I inhibitor. Interestingly, IL-I activity was twice normal in this patient whereas all other patients had normal or lower levels of IL-I. Studies of additional scleroderma patients on d-penicillamine is needed to determine if these changes represent a biological effect of this drug in scleroderma. When indomethacin was added to in-vitro cultures of normal PBMC, there was a marked decrease in IL-1 inhibitor production (Berman et al., 1986). Decreased production of IL-I inhibitor was also seen when indomethacin or aspirin was given to normal volunteers (unpublished observation). However, in the four scleroderma patients taking nonsteroidal antiinflammatory drugs, these drugs were not effective in lowering the high levels of the IL-I inhibitor. No patients were taking corticosteroids and the effects of slow channel calcium blocking agents (nifedipine) and cimetidine on IL-1 and IL-1 inhibitor production are unknown. Whether the study of circulating monocytes may reflect actual changes in the fibroblast microenvironment is unknown. Duncan, Perlish & Fleischmajer (1984) described a concomitant decrease in the production of a fibroblast growth inhibitory lymphokine and increase in the production of a fibroblast inhibitory monokine from scleroderma peripheral blood mononuclear cells and proposed that these changes could represent regulatory responses occurring during the disease. Scleroderma sera are mitogenic for fibroblasts (Potter et al., 1984; Kahaleh & LeRoy, 1986). Invitro studies have shown that scleroderma sera may lead over time to selection of populations of fibroblasts which produce high amounts of collagen (Botstein, Sherer & LeRoy, 1982). To determine if the IL-1 inhibitor with fibroblast-stimulating activity that we have defined plays an important role in the pathogenesis of scleroderma, we will need to demonstrate increased production of the inhibitor by tissue macrophages or cellular infiltrates and/or increased levels of the IL-I inhibitor in scleroderma sera or in the tissues. As knowledge of the basic biological processes involved in scleroderma is gathered, it becomes
IL-] inhibitor with fibroblast stimulating activity
3I9
increasingly apparent that cells of the immune system are involved in the pathogenesis. These cells and the factors they produce may be responsible not only for increased fibroblast proliferation and collagen synthesis but also for the endothelial damage and other immune abnormalities noted in this disease. We wish to thank Susan Burns and Linda Walter for their assistance in preparing the manuscript. This work was supported by NIH grant No. AM30876 and Arthritis Foundation Institution Grants, Southern California Chapter.
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response to
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progressive systemic sclerosis patients: role of suppressor monocytes. Clin. exp. Immunol. 47, 147. YAM, J.T., LI, C.Y. & CROSBY, W.H. (1971) Cytochemical identification of monocytes and granulocytes. Am. J. clin. Pathol. 55, 283.
