TGF-β in blood: a complex problem

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Abstract

The cytokine transforming growth factor-β (TGF-β) was initially purified from human platelets, a rich source of this protein. In addition to platelets, TGF-β1 is also found in other blood fractions, including plasma and the circulating leukocytes. However, more than 15 years after the initial isolation of TGF-β1, there remains no consensus on how much TGF-β1 is present in normal human plasma. Here we review the difficulties associated with measuring TGF-β concentrations in complex biological fluids, and discuss the current state of knowledge on the distribution of TGF-β isoforms in various blood fractions as well as the nature of the TGF-β-containing protein complexes.

Introduction

The transforming growth factor-β (TGF-β) superfamily is a collection of structurally related multi-functional cytokines that have been implicated in a wide range of physiological and pathological processes, including wound healing, development, oncogenesis, immunomodulation and atherosclerosis [1]. The prototypical member of this superfamily, TGF-β1, was originally identified as a growth factor for transformed cells, but was first purified to homogeneity from human platelets [2]. There are now known to be three closely related mammalian TGF-β isoforms (TGF-β1, -β2 and -β3) which are thought to have similar functions, at least in vitro, although less is known about TGF-β2 and TGF-β3. Each of the three isoforms is produced as a pre-pro-protein which rapidly dimerises. After loss of the signal sequence the dimer is further processed by addition of sugar moieties to the propeptide region (known as the latency-associated peptide; LAP). In addition, there is proteolytic cleavage between the LAPs and the mature dimer (which, when released from the LAP dimer, is able to bind to the TGF-β signalling receptors). After cleavage, however, the LAP dimer usually remains non-covalently associated with the mature dimer forming a complex known as the small latent complex [3]. Either prior to secretion or in the extracellular milieu the small latent complex can bind to a wide range of other proteins forming a large number of higher molecular weight latent complexes. The best characterised of these proteins are the latent TGF-β binding protein family (LTBP1-4 and fibrillin-1 and 2) [4], [5]. For example, in platelets, the majority of the TGF-β present is thought to exist in a latent complex consisting of the LTBP-1 covalently coupled to the small latent complex. This complex is called the large latent complex.

Once in the extracellular environment (such as the extracellular matrix of solid tissues or in the blood plasma), the latent complexes, possibly associated with various accessory proteins, must be activated in order to exert their biological effects. It is assumed that this activation process involves release of the mature dimer from its association with the LAP dimer. A number of pathways are likely to participate in TGF-β activation, such as cleavage of the LAP dimer by proteases, including plasmin [6], [7], and conformational changes in the latent complex induced by binding to matrix components such as thrombospondin [8], [9] or certain integrins [10]. Once activated, the TGF-β is able to signal through cell surface receptors, of which the best studied are the type I and type II TGF-β receptors (TβRI and TβRII) which can form heteromultimers in response to ligand binding resulting in an active signalling complex which is thought to be responsible for many of the cellular responses to TGF-β [11].

TGF-β1 has a very wide range of activities in vitro. For example, TGF-β regulates important cellular functions such as rate of proliferation and production of extracellular matrix proteins by a wide range of cell types [1]. Thus, misregulation of TGF-β has been proposed to play a key role in the development of a number of diseases in which the normal adult tissue architecture is progressively lost, including scarring during wound repair [12], carcinogenesis [13], atherosclerosis [14], osteoporosis [15], [16] and neurodegenerative diseases [17].

As a result of the wide range of activities attributed to TGF-β, a number of groups have investigated whether circulating levels of TGF-β1 might be altered in various disease states. With only one exception, all of these studies agree that TGF-β1 is found at detectable levels in plasma from healthy human subjects (see Table 1). Moreover, plasma TGF-β1 concentrations markedly differed (by as much as 10-fold) in subjects suffering from various diseases, including various cancers [18], [19], [20], [21], autoimmune disorders [22], [23] and atherosclerosis [24], [25], compared with control subjects. Based on these early studies, it seems likely that plasma levels of TGF-β1 may be a useful diagnostic criteria for the presence of one or more of these dieases. In addition, it remains possible, but unproven, that altered plasma levels of this multifunctional cytokine participates in disease progression, rather than simply acting as a marker for disease status. If such a pathophysiological role for plasma TGF-β1 is proven, it could become both a prognostic indicator of future risk of disease and a target for therapeutic intervention.

Section snippets

Levels of TGF-β in normal human plasma

Despite the interest in measuring levels of TGF-β1 in plasma as a result of early reports suggesting associations between plasma TGF-β1 concentration and disease, there is still no consensus on the concentration range of TGF-β1 in normal human plasma. The levels of TGF-β1 protein in plasma from various groups of normal subjects have been measured in more than 20 studies in the literature to date, yet the mean (or median) values reported range from below 0.1 ng/ml to more than 25 ng/ml (Table 1).

Initial characterisation of the TGF-β1 complexes in human plasma

We have recently used column chromatography to begin to address the question of the molecular composition of the TGF-β1 complexes in plasma [36]. When plasma, serum or platelets are applied to an anion exchange column and then unbound material washed away, all the TGF-β1 is retained on the column (assessed using the Quantikine TGF-β1 assay). Any TGF-β1 complexes which are not detected by the Quantikine TGF-β1 ELISA are, by definition, excluded from this analysis. The bound TGF-β1 can then be

Where does plasma TGF-β come from?

There are a number of potential sources for the TGF-β1 present in normal human plasma. Although release of platelet TGF-β1 during blood sampling and plasma preparation can be excluded as an artefactual source of plasma TGF-β1, it remains possible that platelets are a source of plasma TGF-β1 in vivo, either through regulated secretion or a background level of platelet degranulation. However, comparison of the column retention times of the plasma TGF-β1 complexes with that of the platelet TGF-β1

What is the mechanism of clearance of TGF-β1 from the plasma?

When radiolabelled TGF-β1 25 kDa dimer is injected into the blood, it is rapidly lost from the circulation (half-life=∼2 min; [43], [44], [45]). Analysis of the distribution of the radioactive label demonstrated that it becomes localised to the vascular endothelium [46], possibly as a result of binding to the abundant type III TGF-β receptor, endoglin (CD105). One methodological consequence of this rapid association with the endothelium is that it is difficult to analyse any other routes of

What controls the levels of plasma TGF-β?

At present there is little quantitative data to indicate to what extent regulated synthesis or regulated clearance contribute to the control of the steady state level of plasma TGF-β1. However, we have recently obtained some indirect evidence that synthesis rate plays a part in determining steady state plasma levels of TGF-β1 protein, at least in healthy individuals. Analysis of a population of post-menopausal female twins (comparing monozygotic with dizygotic pairs) has demonstrated that

Is there any active TGF-β in normal human plasma?

A number of assays, both bioassays and ELISAs, have been described which attempt to measure the amount of active TGF-β1 in complex biological fluids, such as plasma. In some cases, assays which previously have been used to measure total TGF-β1 protein after in vitro activation of the sample (such as by transient acidification), have also been used to measure active TGF-β in the sample prior to activation. There is one key advantage to using bioassays for measuring active TGF-β1 — bioassays

Conclusions

The variability among reported levels of TGF-β1 in human plasma stems primarily from two sources — preparation of plasma samples and selection of assay methodology. Appropriate protocols are now available [26] to ensure that plasma can be prepared with minimal contamination from other blood fractions, such as platelets, and widespread adoption of these protocols should reduce the variation in values reported in different studies. However, the variability due to selection of assay methodology

References (81)

  • M.A van Waarde et al.

    Quantification of transforming growth factor-beta in biological material using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct

    Anal. Biochem.

    (1997)
  • D.J Grainger et al.

    Active and acid-activatable TGF-beta in human sera, platelets and plasma

    Clinica Chimica Acta

    (1995)
  • N Nishimura et al.

    Acquisition of secretion of transforming growth factor-beta 1 leads to autonomous suppression of scavenger receptor activity in a monocyte-macrophage cell line, THP-1

    J. Biol. Chem.

    (1998)
  • A Philip et al.

    Interaction of transforming growth factor-beta 1 with alpha 2-macroglobulin. Role in transforming growth factor-beta 1 clearance

    J. Biol. Chem.

    (1991)
  • U Junker et al.

    Transforming growth factor beta 1 is significantly elevated in plasma of patients suffering from renal cell carcinoma

    Cytokine

    (1996)
  • M Shah et al.

    Role of elevated plasma transforming growth factor-beta1 levels in wound healing

    Am. J. Pathol.

    (1999)
  • F Muller et al.

    Reduced serum level of transforming growth factor-beta in patients with IgA deficiency

    Clin. Immunol. Immunopathol.

    (1995)
  • A Hayasaka et al.

    Plasma levels of transforming growth factor beta 1 in chronic liver disease

    Clin. Chim. Acta

    (1996)
  • T Murase et al.

    Transforming growth factor-beta plasma concentrations in patients with leukemia and lymphoma receiving chemoradiotherapy and marrow transplantation

    Blood

    (1994)
  • C.H Szymkowiak et al.

    Determination of transforming growth factor beta 2 in human blood samples by ELISA

    J. Immunol. Methods

    (1995)
  • C.H Szymkowiak et al.

    Determination of transforming growth factor beta 2 in human blood samples by ELISA

    J. Immunol. Methods

    (1995)
  • A.M Gray et al.

    Requirement for activin A and transforming growth factor-beta 1 pro-regions in homodimer assembly

    Science

    (1990)
  • K Miyazono et al.

    A role of the latent TGF-beta-1-binding protein in the assembly and secretion of TGF-beta-1

    Embo Journal

    (1991)
  • P.A Dennis et al.

    Cellular activation of latent transforming growth-factor-beta requires binding to the cation-independent mannose 6-phosphate insulin-like growth-factor type-ii receptor

    Proc. Natl. Acad. Sci., USA

    (1991)
  • S Schultz-Cherry et al.

    Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism

    J. Cell Biol.

    (1993)
  • J Massagué

    TGF-beta signal transduction

    Annu. Rev. Biochem.

    (1998)
  • W.A Border et al.

    Transforming growth factor-beta in disease: the dark side of tissue repair

    Journal of Clinical Investigation

    (1992)
  • R.J Akhurst et al.

    Genetic events and the role of TGF beta in epithelial tumour progression

    J. Pathol.

    (1999)
  • D.J Grainger et al.

    A pivotal role for TGF-beta in atherogenesis?

    Biological Reviews of the Cambridge Philosophical Society

    (1995)
  • T Fujita et al.

    Cytokines and osteoporosis

    Ann. NY Acad. Sci.

    (1990)
  • D.J Grainger et al.

    The role of serum TGF-beta isoforms as potential markers of osteoporosis

    Osteoporos Int.

    (1999)
  • Y Shirai et al.

    Plasma transforming growth factor-beta 1 in patients with hepatocellular carcinoma. Comparison with chronic liver diseases

    Cancer

    (1994)
  • V Ivanovic et al.

    Elevated plasma levels of TGF-beta 1 in patients with invasive prostate cancer

    Nat. Med.

    (1995)
  • K Krasagakis et al.

    Elevated plasma levels of transforming growth factor (TGF)-beta1 and TGF-beta2 in patients with disseminated malignant melanoma

    Br. J. Cancer

    (1998)
  • F.M Kong et al.

    Elevated plasma transforming growth factor-beta 1 levels in breast cancer patients decrease after surgical removal of the tumor

    Ann. Surg.

    (1995)
  • A Pfeiffer et al.

    Elevated plasma levels of transforming growth factor-beta 1 in NIDDM

    Diabetes Care

    (1996)
  • N Snowden et al.

    Plasma TGF beta in systemic sclerosis: a cross-sectional study

    Ann. Rheum. Dis.

    (1994)
  • D.J Grainger et al.

    The serum concentration of active transforming growth-factor-beta is severely depressed in advanced atherosclerosis

    Nature Medicine

    (1995)
  • X.L Wang et al.

    Circulating transforming growth factor beta 1 and coronary artery disease

    Cardiovascular Research

    (1997)
  • L.M Wakefield et al.

    Transforming growth factor-beta1 circulates in normal human plasma and is unchanged in advanced metastatic breast cancer

    Clin. Cancer Res.

    (1995)
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