Active and inhibitory components of the insulin-like growth factor binding protein-3 protease system in adult serum, interstitial, and synovial fluid.

Circulating insulin-like growth factor binding protein-3 (IGFBP-3) proteolytic activity is normally low but increases in serum from pregnant women and from patients with various pathologies. In contrast, we have recently reported that outside the circulation, such activity is normally high but decreases in various pathologies. We have now compared components of the IGFBP-3 proteolytic system revealed after size fractionation of serum and extravascular fluids with different intrinsic levels of such activity. Normal serum, serum from pregnant women, and synovial fluid from patients with rheumatoid arthritis revealed high and low molecular weight (MW) areas of activity. However, only the low MW activity was apparent in interstitial fluid from normal skin (N Inst F) or psoriatic lesions (P Inst F) and in synovial fluid from normal volunteers (N Syn F) or patients with osteoarthritis (OA Syn F). Addition of inhibitors revealed both areas to comprise more than one enzyme, including serine proteases and metalloproteinases; both could also be inhibited by P Inst F, NS, RA Syn F, and inhibitory fractions from the separation of the latter two. These findings demonstrate low and high MW regions of proteolytic activity, which may contribute to the IGFBP-3 protease system, the former always present, whereas the latter seems to be retained within the circulation apart from inflammatory conditions. The variations apparent in IGFBP-3 protease activity in the intact samples related to the presence of an inhibitor, which may protect IGFBP-3 from proteolysis, rather than to changes in the component proteases.

Ethnicity affects IGFBP-3 and IGF-II in normal healthy young adult subjects.

OBJECTIVE: While the effects of age on the growth hormone/insulin-like growth factor (IGF) axis are well documented, the influence of ethnic background is unknown. The differences in IGF and IGF binding proteins (IGFBPs) were investigated in two ethnic groups. DESIGN: A cross-sectional study of an age-selected cohort of healthy, normoglycaemic, non-obese Caucasian (C) and Asian (A) subjects. PATIENTS: Fifty-three (27 C, 26 A) subjects with a mean age (+/- SD) of 20.6 +/- 0.8 years were studied. MEASUREMENTS: Fasting measurements of glucose, insulin, IGF-I, IGF-II, IGFBP-1 and IGFBP-3. Western ligand blotting and immunoblotting with IGFBP-2 and IGFBP-3 of serum samples. RESULTS: There were no significant differences in IGF-I levels between Caucasian and Asian subjects (C 218 +/- 55 vs A 229 +/- 40 micrograms/l; P = 0.44). IGF-II (C 707 +/- 110 vs A 583 +/- 75 micrograms/l; P < 0.0001) and IGFBP-3 (C 5.9 +/- 1.2 vs A 5.12 +/- 1.17 mg/l; P = 0.01) levels were significantly higher in Caucasian subjects. Immunoblotting of ligand blots revealed no protease activity on either IGFBP-3 or IGFBP-2 to account for these ethnic differences. CONCLUSIONS: Ethnic differences in IGFBP-3 and associated IGF-II levels may affect the inter-relationships of IGFs and their binding proteins and need to be considered when interpreting IGF data on growth and metabolism.

Insulin-like growth factor (IGF) I and II, IGF-binding proteins, and IGF-binding protein proteases are produced by theca and stroma of normal and polycystic human ovaries.

There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovarian tissues. It has been suggested that abnormalities of intraovarian IGF-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I and -II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatography in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in the low nanograms per mL range in theca- and stroma-conditioned medium (T+SCM). IGFBPs in T+SCM were initially analyzed by Western ligand blotting, which revealed that low mol wt IGFBPs were predominant, especially IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with smaller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences between normal and polycystic ovary syndrome ovaries, and although there was a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Western ligand blotting in granulosa cell-conditioned medium. In further studies we attempted to measure IGFBP-3 by RIA using two different antisera (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFBP-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287-2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no evidence of proteolysis of serum IGFBP-3 after incubation with conditioned medium, but in contrast, radiolabeled [125I]IGFBP-3 was cleaved after incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive fragments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM contained IGF-I and -II. IGFBP-2 and -4 were the predominant species of IGFBP in T+SCM. T+SCM also contained significant protease activity directed toward IGFBP-2 and -3. Proteolytic activity may be an important mechanism by which bioactive IGFs are made available to these tissues.

Altered insulin-like growth factor-II (IGF-II) level and IGF-binding protein-3 (IGFBP-3) protease activity in interstitial fluid taken from the skin lesion of psoriasis.

In the present study, we have investigated insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in serum and artificially raised blister fluid from uninvolved and involved areas of nine patients with psoriasis. Both levels of IGFs and IGFBP-3, and profiles of IGFBP in serum and fluid from the uninvolved areas of these patients were comparable to those seen in normal subjects. In fluid from the involved areas, the IGF-II but not IGF-I level was significantly elevated. Among five molecular forms of IGFBP, the density of 41.5- and 38.5-kDa forms of IGFBP-3 were apparently increased in fluid from the involved areas, shown by Western ligand blotting. Radioimmunoassay further showed that the IGFBP-3 concentration in the involved areas was significantly raised. Immunoblotting revealed that the predominant form of IGFBP-3 in fluid from the uninvolved areas was a 29-kDa proteolytically modified product. In contrast, intact doublet IGFBP-3 was the main form of IGFBP-3 in fluid from the involved areas. Fluid from the involved areas but not the matched serum concentration-dependently inhibited the degradation of 125I-labeled nonglycosylated IGFBP-3 (ngIGFBP-3) caused by fluid from the uninvolved areas, suggesting the presence of an IGFBP-3 protease inhibitor(s) in psoriatic skin lesion. These findings suggest that the alterations in IGF/IGFBP system may contribute to the pathogenesis of psoriasis.

Insulin-like growth factors (IGFs) and IGF-binding proteins in human skin interstitial fluid.

Despite extensive investigation of the insulin-like growth factor (IGF)/IGF-binding protein (IGFBP) system in the circulation and body fluids, there is no information on this in interstitial fluid. We have compared the IGF/IGFBP system in the circulation with that in fluid obtained from blisters artificially raised by negative pressure in 10 healthy volunteers. IGFBP-1, -2, -3, and -4 were all found in blister fluid, but in concentrations much lower than those in matched serum. The IGF-I, IGF-II, and IGFBP-3 levels measured by RIA were 18%, 14%, and 16% of those in serum, respectively. Fast protein liquid chromoatography showed that both IGF-I and IGFBP-3 in 150- and 50-kilodalton complexes were approximately 13% and 37%, respectively, of the corresponding peaks found in matched serum. Compared to that in serum, the IGFBP-3 in the blister fluid was predominantly in a modified 29-kilodalton form, and there was increased activity of an IGFBP-3 protease. Therefore, although IGF concentrations are much lower in interstitial fluid than in the circulation, a greater proportion of this IGF is in forms more readily available for interaction with tissue receptors. The blister fluid appears to represent physiological interstitial fluid and may provide a model for studying the physiology and pathophysiology of growth factors in the interstitial environment.

Two site-specific radioimmunoassays which demonstrate the presence of proteolytically modified insulin-like growth factor-binding protein-3 in the circulation.

The presence of proteolytic activity in the circulation directed against insulin-like growth factor binding protein-3 (IGFBP-3) was originally described in pregnancy but has subsequently been described in a number of catabolic and other pathological conditions. However, detection of this proteolytically modified IGFBP-3 has been demonstrated, in most cases, following separation using SDS-PAGE and this has led to speculation that its occurrence may be an artefact of the harsh conditions employed. Using two site-specific antisera, one of which recognizes as its antigenic site a region of IGFBP-3 which is close to that of the IGF-binding domain, we have developed two radioimmunoassays which, when compared, can reveal alterations in the IGF-binding domain of IGFBP-3. The presence of IGFBP-3 modified by the circulating protease is denoted in this system by a divergence in the IGFBP-3 levels observed; this divergence has been shown to coincide with protease activity as determined by Western ligand blotting. The use of these assays has confirmed that the IGF-binding site of IGFBP-3 undergoes proteolytic modification in the circulation during a number of pathologies.

Measurement of insulin-like growth factor-II in human plasma using a specific monoclonal antibody-based two-site immunoradiometric assay.

An immunoradiometric assay (IRMA) for the measurement of insulin-like growth factor-II (IGF-II) in human plasma has been developed, optimized and evaluated clinically in normal subjects and patients with disorders of the GH/IGF-I axis. Six monoclonal antibodies (MAbs) to recombinant human IGF-II (rhIGF-II) were produced, all of which had low cross-reactivity with rhIGF-I (< 0.01%) and insulin (< 0.01%). Compatibility of pairs of MAbs was tested in two-site IRMAs using three radioiodinated MAbs and three MAbs linked to Sephacryl S-300 (with separation of bound and free radiolabelled MAb by sucrose layering). Seven pairs of MAbs bound rhIGF-II and the combination of 125I-labelled W3D9 and W2H1 linked to solid phase was selected. The optimized assay had a completion time of 4 h, a minimum detection limit of 30 ng/ml (2.5 standard deviations from the zero standard) and detected a single peak of endogenous IGF-II in normal plasma which co-eluted with rhIGF-II after acid gel chromatography. IGF-II was measured in formic acid/acetone extracts of plasma from 16 normal subjects (mean 685, range 516-1008 micrograms/l), four acromegalic patients (mean 637, range 553-700 micrograms/l), fourteen patients with type-1 diabetes (mean 635, range 247-753 micrograms/l), nine patients with uraemia (mean 423, range 78-850 micrograms/l), and three patients with Laron-type GH insensitivity (75, 35 and 36 micrograms/l). No significant fluctuations were detected between samples obtained hourly from 08.00 to 19.00 h in normal subjects. Low levels of IGF-binding proteins (IGFBPs) remaining in plasma extracts may interfere with the measurement of IGF-II and give rise to falsely elevated IGF-II levels in radioimmunoassays or falsely suppressed levels in IRMAs. Such interference did not occur with the IRMA when used to measure IGF-II in extracts from normal subjects, acromegalic patients and patients with type-1 diabetes, and the addition of excess rhIGF-I in order to displace IGF-II from residual IGFBPs had no effect on IGF-II measurements in these samples. However, levels of IGF-II measured in extracts from patients with Laron-type GH insensitivity and patients with uraemia increased markedly after preincubation with excess rhIGF-I. The accurate measurement of IGF-II by IRMA in extracts from these subjects therefore requires the displacement of IGF-II from IGFBPs prior to assay. We conclude that, in contrast to radioimmunoassays, the two-site IRMA developed here provides a practical, rapid and specific method for the measurement of IGF-II in human plasma.

Cytokines modulate the sensitivity of human fibroblasts to stimulation with insulin-like growth factor-I (IGF-I) by altering endogenous IGF-binding protein production.

Human dermal fibroblasts produce a number of insulin-like growth factor-binding proteins (IGFBPs) including the main circulating form, IGFBP-3. It has been suggested that the regulation of IGFBP secretion may play a major role in modulating insulin-like growth factor (IGF) bioactivity. We have quantified the effects of two cytokines, transforming growth factor-beta 1 (TGF-beta 1) and tumour necrosis factor-alpha (TNF-alpha) which have opposing actions on fibroblast IGFBP-3 production, and examined their subsequent role in IGF-I mitogenesis. TGF-beta 1 caused a dose-dependent increase in IGFBP-3 in serum-free fibroblast-conditioned media. TGF-beta 1 (1 microgram/l) resulted in immunoreactive IGFBP-3 levels reaching 286.5 +/- 22.4% of control after 20 h, the increase being confirmed by Western ligand blot. TNF-alpha caused a dose-dependent decrease in fibroblast IGFBP-3 secretion, 1 microgram TNF-alpha/l reducing IGFBP-3 levels to 32.1 +/- 11.% of control. This effect was not due to cytotoxicity and was not cell-density-dependent. Fibroblast proliferation was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric cytochemical bioassay. The addition of IGF-I resulted in dose-dependent growth stimulation after 48 h, the effective range being 20-100 micrograms/l. The IGF-I analogue Long-R3-IGF-I which has little affinity for the IGFBPs was approximately 20-fold more potent in this assay, and was unaffected by exogenous IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of insulin-like growth factor-binding proteins on steroidogenesis by human granulosa cells in culture.

The effects of insulin-like growth factor-binding proteins (IGFBPs) 1 and 3 on steroidogenesis by human granulosa cells has been examined. Both IGFBP-1 and IGFBP-3 produced a dose-related inhibition of IGF-I-stimulated oestradiol accumulation in granulosa cell-conditioned medium with complete reversal of the effects of IGF-I in the presence of a molar excess of binding protein. IGFBPs 1 and 3 also exerted a small (25-40%) but significant and consistent inhibition of oestradiol secretion in response to follicle-stimulating hormone (FSH) alone. The progesterone response to IGF-I was inhibited by IGFBPs 1 and 3 but there was no effect on FSH-stimulated progesterone production. These data support the concept of a physiologically important intraovarian IGF system in the human ovary and demonstrate an unequivocally inhibitory effect of IGFBPs 1 and 3 on IGF-I-stimulated granulosa cell steroidogenesis.

The induction of specific proteases for insulin-like growth factor-binding proteins following major heart surgery.

Insulin-like growth factors (IGF-I and IGF-II) circulate bound to specific high-affinity binding proteins (IGFBPs). Recent evidence has shown that in pregnancy and severe illness, specific proteases modify these binding proteins, reducing their affinity for IGFs. We have studied 12 patients, undergoing elective coronary artery vein-bypass graft surgery, for the appearance of these proteases and have demonstrated the induction of two independent, heat-labile, cation-dependent proteases. Proteolytic activity directed against IGFBP-3 was detected in all patients between 24 h and 5 days after surgery; the second IGFBP-4 specific protease was active 1 h after sternotomy. The total IGF-I levels were found to decrease following surgery, with the IGF-I distribution in the plasma being radically altered from that seen prior to the operation. One day after the operation the majority of the IGF-I, instead of being bound in the relatively inert 150 kDa complex, was associated with the smaller binding proteins which are more readily accessible to the tissues. These findings are in contrast to pregnancy where, despite similar proteases, the majority of the IGF-I remains in the 150 kDa complex. The alteration seen in IGF-I distribution after surgery did not appear to be a direct result of the IGFBP-3 proteolytic activity or an effect of the addition of heparin to the circulation. The potential increase in bioavailability of IGFs caused by the alteration in carrier protein may play a pivotal role in countering the catabolic state induced by surgery.