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. 1999 Mar;115(3):409–414. doi: 10.1046/j.1365-2249.1999.00839.x

Evidence for control of tumour necrosis factor-alpha (TNF-α) activity by TNF receptors in patients with proliferative diabetic retinopathy

G A Limb 1, H Soomro 1, S Janikoun 1, R D Hollifield 1, J Shilling 1
PMCID: PMC1905243  PMID: 10193411

Abstract

TNF-α has been implicated in the pathogenesis of insulin- dependent diabetes mellitus (IDDM). At present there are no studies linking serum levels of soluble TNF receptors (sTNF-R) to the development of diabetic microvascular complications such as proliferative diabetic retinopathy (PDR), or to the production of TNF-α in these patients. We investigated serum levels of sTNF receptors (sTNF-RI and sTNF-RII) in IDDM patients with or without PDR, and related these to the in vitro production of TNF-α upon activation of whole blood and isolated mononuclear cells (MNC). We observed higher serum levels of sTNF-RI in IDDM patients with active (range 945–6630 pg/ml; P = 0.029) or quiescent PDR (range 1675–4970 pg/ml; P = 0.00092) than in individuals with IDDM without retinopathy (range 657–2617 pg/ml) or healthy controls (range 710–1819 pg/ml; P = 0.0092 and 0.0023, respectively). Increased serum levels of sTNF-RII were also seen in IDDM patients with active PDR (range 1749–5218 pg/ml; P = 0.034) or quiescent PDR (range 1494–5249 pg/ml; P = 0.0084) when compared with disease controls (range 1259–4210 pg/ml) or healthy subjects (range 1237–4283 pg/ml). Whole blood production of biologically active TNF-α was lower in PDR patients than in disease (P = 0.04) and healthy controls (P < 0.005), contrasting with a higher production of TNF-α by lipopolysaccharide (LPS)-activated MNC from PDR patients (P = 0.013). Inhibition of TNF-α by TNF-R in plasma supernatants of activated blood from PDR patients was demonstrated by increase of TNF-α activity in the presence of anti-TNF-RI and anti-TNF-RII antibodies. These observations suggest that abnormalities in TNF-α production and control may operate during the development of microvascular complications of diabetes mellitus.

Keywords: sTNF-R, tumour necrosis factor, insulin-dependent diabetes mellitus, PDR

INTRODUCTION

TNF-α, a cytokine generated upon cellular activation, is known to be angiogenic, fibrogenic, proinflammatory and vasculo-reactive [1,2]. Several lines of evidence have implicated TNF-α in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) [3–6], whilst susceptibility to diabetic retinopathy has been associated with TNF-α gene polymorphism [7] and expression of HLA-DR3 and HLA-DR4 phenotypes [8–10]. Since the genes coding for both TNF-α and HLA-DR antigens are located within the MHC [11,12], it is possible that abnormal production and control of TNF-α may be associated with the MHC-linked pathology observed in IDDM.

Proliferative diabetic retinopathy (PDR), a common microvascular complication of IDDM, is characterized by active neovascularization of the retina and formation of fibrovascular tissue at the vitreoretinal interface [13], which require local production of cell-derived angiogenic factors and synthesis of extracellular matrix components necessary for anchorage of migrating endothelium [14,15]. High serum levels of TNF-α precede and accompany the onset of human IDDM [3], cells expressing mRNA coding for TNF-α are found in the islets of Langerhans of non-obese diabetic (NOD) mice during early stages of the disease [4], injection of TNF-α into female newborn NOD mice accelerates the development of diabetes mellitus [5], and leucocytes from patients with IDDM produce increased levels of TNF-α upon activation in vitro [6]. In addition, TNF-α is found in the extracellular matrix, endothelium and vessel walls of fibrovascular tissue of PDR [16], and in vitreous from eyes with this complication [17,18].

The biological activity of TNF-α may be inhibited in vitro by TNF receptors (TNF-R), known as TNF-RI (55 kD mol. wt) and TNF-RII (75 kD mol. wt) [19,20]. Soluble (s) forms of both receptors are released from TNF-α-producing cells by proteolytic cleavage [21], and significant levels of both molecules are normally present in serum of healthy individuals [22]. However, raised levels of both sTNF-RI and sTNF-RII are often found in pathological conditions, where circulating levels of these molecules correlate with disease activity [23,24].

Despite the above evidence, it is not clear whether development of microvascular complications of IDDM, particularly proliferative diabetic retinopathy, are associated with high serum levels of sTNF-R or abnormal production of TNF-α. On this basis we investigated the serum levels of sTNF-RI and sTNF-RII in patients with IDDM complicated or uncomplicated by PDR, and related these to the levels of production of TNF-α by whole blood stimulated with lipopolysaccharide (LPS), and to the production of TNF by LPS-activated mononuclear cells.

PATIENTS AND METHODS

Fifty-three patients with IDDM attending the diabetic eye and medicine clinics at St Thomas' Hospital were selected for the study upon prior written consent on the basis that their diabetes was of young onset, insulin-dependent, of at least 10 years duration, and that they had either developed proliferative diabetic retinopathy or not presented with any form of retinopathy or other severe microvascular complications. The study was approved by the ethical committee of St Thomas' Hospital. The main clinical characteristics of the individuals entered in the study are summarized in Table 1. Healthy individuals matching sex and age of the patients were used as controls.

Table 1.

Clinical features of patients included in the study

graphic file with name cei0115-0409-t1.jpg

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Assessment of retinopathy

Diabetic individuals included in the study were divided into three main groups: (i) those with no retinopathy, as judged by absence of microaneurisms, macular oedema or hard exudate formation by fundus photography; (ii) those with severe PDR, as judged by new vessel proliferation, severe intraretinal vascular abnormalities, photocoagulation scars and preretinal or vitreous haemorrhages; and (iii) those with quiescent PDR, who had been successfully treated with laser photocoagulation for this condition. Proliferative diabetic retinopathy was confirmed by direct and indirect ophthalmoscopy and slit-lamp biomicroscopy following pupillary dilation [25].

LPS activation of whole blood

Whole heparinized blood (1 ml) was incubated with 100 ng/ml of LPS derived from Escherichia coli (Sigma, Poole, UK). Following 24 h incubation, plasma supernatants from the activated blood, diluted 1:4 with culture medium (RPMI 1640), were harvested and kept at −70°C until use.

Determination of serum sTNF-RI and sTNF-RII

Serum from 42 individuals with IDDM and 20 normal subjects were investigated for the levels of immunoreactive sTNF-R by ELISA using commercially available kits (R&D Systems, Abingdon, UK) and our published methods [26].

Determination of biologically active TNF in whole blood stimulated with LPS

Biologically active TNF was measured in plasma supernatants of LPS-activated blood from 37 IDDM patients and 16 controls by a cytotoxic assay using WEHI-1 cells [17]. Briefly, WEHI-1 cells (2.5 × 105/ml) were cultured with serial dilutions of plasma in the presence of actinomycin D (final concentration 1 μg/ml). After 24 h incubation, the proportion of viable cells was determined by a colorimetric assay based on the reduction of MTS (Promega, Southampton, UK). Levels of TNF present in the plasma supernatants were interpolated from specific calibration curves prepared with known standard solutions (National Institute for Biological Standards and Control, South Mimms, UK).

To determine whether sTNF-R in plasma of LPS-activated blood from PDR patients were responsible for the decrease in TNF-α levels compared with healthy controls, plasma supernatants from two individuals with PDR were preincubated at 37°C for 4 h with anti-TNF-RI and anti-TNF-RII antibodies (Hycult Biotechnology b.v., Loughborough, UK) at a final concentration of 25 μg/ml. Levels of biologically active TNF-α were then determined as described above.

Production of immunoreactive TNF-α by whole blood and mononuclear cells upon LPS stimulation

Mononuclear cells (MNC) from 38 patients and 11 controls were isolated from whole blood by gradient sedimentation on Lymphoprep (Nycomed Pharma AS, Oslo, Norway). MNC were cultured (1 × 106 cells/ml) for 24 h at 37°C in RPMI medium containing 5% fetal calf serum (FCS) and 1 ng/ml LPS. Cell preparations were centrifuged at 400 g and the cell-free supernatants removed and stored at −70°C until use. Immunoassays for measurement of TNF-α in all MNC supernatants and plasma of LPS-activated blood from a small number of individuals (seven from each of the groups under study) were performed by immunoassays using commercially available kits (R&D Systems) as described above for sTNF-R.

RESULTS

Serum levels of sTNF-RI and sTNF-RII in IDDM patients complicated or uncomplicated by PDR; comparison with healthy subjects

As seen in Fig. 1, serum levels of sTNF-RI in IDDM patients complicated by PDR were significantly higher (range 945–6630 pg/ml) than in IDDM patients without PDR (range 657–2617 pg/ml: Mann–Whitney test, P = 0.029; Fig. 1) and healthy individuals (range 710–1819 pg/ml: Mann–Whitney test, P = 0.009). Serum levels of sTNF-RI in IDDM patients with quiescent PDR were also significantly higher (range 1675–4970 pg/ml) than in patients without PDR (Mann–Whitney test, P = 0.0009) or healthy individuals (Mann–Whitney test, P = 0.0002) (Fig. 1). Similarly, serum levels of sTNF-RII were markedly elevated (range 1749–5218 pg/ml) in patients with IDDM complicated by PDR when compared with normal subjects (range 1237–4283 pg/ml; Mann–Whitney test, P = 0.035; Fig. 1) or individuals with IDDM without PDR (range 1259–4210 pg/ml; Mann–Whitney test, P = 0.034). Serum levels of sTNF-RII in patients with quiescent PDR were also higher (range 1494–5249 pg/ml) than in individuals with uncomplicated IDDM (Mann–Whitney test, P = 0.0084) or healthy controls (Mann–Whitney test, P = 0.015; Fig. 1).

Fig. 1.

Fig. 1

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Comparison between serum levels of sTNF-RI and sTNF-RII in patients with insulin-dependent diabetes mellitus (IDDM) complicated or uncomplicated by proliferative diabetic retinopathy (PDR) and healthy controls. Mann–Whitney U-tests: *P = 0.029 and 0.0092 (versus IDDM no PDR and healthy controls, respectively); **P = 0.000 92 and 0.0023 (versus IDDM no PDR and healthy controls, respectively); ***P = 0.034 and 0.035 (versus IDDM no PDR and healthy controls, respectively); ****P = 0.0084 and 0.035 (versus IDDM no PDR and healthy controls, respectively). Numbers in parentheses indicate the number of patients included in each group.

Production of biologically active TNF in whole blood of IDDM patients complicated or uncomplicated by PDR and healthy subjects

Figure 2 shows that plasma supernatants from LPS-stimulated blood of IDDM subjects with PDR contained significantly lower levels of biologically active TNF (range 1664–9460 pg/ml) than those of IDDM patients without retinopathy (range 828–18 836 pg/ml; Mann–Whitney test, P = 0.04) or healthy controls matching age and sex of the patients (range 3056–17 620 pg/ml; Mann–Whitney test, P < 0.001).

Fig. 2.

Fig. 2

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Levels of bioactive TNF in plasma supernatants of whole blood activated with lipopolysaccharide (LPS). Comparison between insulin-dependent diabetes mellitus (IDDM) patients complicated or uncomplicated by proliferative diabetic retinopathy (PDR) and IDDM patients without retinopathy or healthy subjects. Mann–Whitney U-tests: *P = 0.04 (versus IDDM no PDR) and P < 0.001 (versus healthy controls); **P < 0.005 (versus IDDM no PDR and healthy controls). The lines represent the median of the TNF-α levels in each group. Numbers in parentheses indicate the number of patients included in each group.

Levels of immunoreactive TNF in whole blood of IDDM patients complicated or uncomplicated by PDR and healthy subjects

Figure 3 shows that similar to levels of biologically active TNF-α, plasma supernatants from LPS-stimulated blood of IDDM subjects with PDR contained significantly lower levels of immunoreactive TNF-α (range 584–1612 pg/ml) than those of IDDM patients without retinopathy (range 696–2968 pg/ml; Mann–Whitney test, P = 0.040) or healthy controls matching age and sex of the patients (range 1560–3120 pg/ml; Mann–Whitney test, P = 0.0049). Despite the differences between the levels of immunoreactive and biologically active TNF observed, the variation in immunoreactive TNF production amongst the groups of patients and controls was consistent with that observed with the production of biologically active TNF-α (Fig. 2).

Fig. 3.

Fig. 3

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Levels of immunoreactive TNF-α in plasma supernatants of lipopolysaccharide (LPS)-activated blood. Comparison between patients with proliferative diabetic retinopathy (PDR), insulin-dependent diabetes mellitus (IDDM) individuals without retinopathy and healthy subjects. Mann–Whitney U-tests: *P = 0.0049 (versus healthy controls); **P = 0.040 (versus IDDM no PDR). The lines represent the median of the TNF-α levels in each group.

Production of TNF-α by LPS-stimulated MNC from IDDM patients complicated or uncomplicated by PDR

As seen in Fig. 4, LPS-activated MNC from IDDM patients with PDR produced significantly higher levels of TNF-α (range 186–1794 pg/106 cells) than MNC from healthy subjects (range 84–482 pg/106 cells; Mann–Whitney test, P = 0.013). There was no difference in the production of this cytokine by MNC from individuals with uncomplicated IDDM (range 67–2726 pg/106 cells) when compared with MNC from patients with PDR (Mann–Whitney test, P = 0.28) or healthy individuals (Mann–Whitney test, P = 0.25).

Fig. 4.

Fig. 4

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Levels of TNF in tissue culture supernatants of isolated mononuclear cells activated with lipopolysaccharide (LPS). Comparison between insulin-dependent diabetes mellitus (IDDM) patients complicated or uncomplicated by proliferative diabetic retinopathy (PDR) and IDDM patients without retinopathy or healthy subjects. Mann–Whitney U-test: *P = 0.013 (versus healthy controls), P = 0.28 (versus IDDM patients without retinopathy). The lines represent the median of the TNF-α levels in each group. Numbers in parentheses indicate the number of patients included in each group.

Inhibition of sTNF-R by antibodies to TNF-RI and TNF-RII in plasma supernatants of LPS-activated blood

Preincubation of plasma supernatants of activated blood from PDR patients with anti-TNF-RI and anti-TNF-RII antibodies caused an increase in their levels of biologically active TNF-α, indicating that high levels of sTNF-Rs may be responsible for the inhibition of TNF activity in the activated plasma from PDR patients when compared with healthy controls or individuals with IDDM without PDR (Fig. 5).

Fig. 5.

Fig. 5

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Inhibition of sTNF-R activity by antibodies to TNF-RI and TNF-RII. Preincubation of plasma from lipopolysaccharide (LPS)-activated blood of proliferative diabetic retinopathy (PDR) patients with anti-TNF-R antibodies (2 μg/ml final concentration) caused an increase in the levels of biologically active TNF-α.

DISCUSSION

A key finding of this study is that IDDM patients with active or quiescent PDR exhibit significantly higher serum levels of sTNF-RI and sTNF-RII than IDDM individuals without retinopathy or healthy subjects. Significantly lower levels of biologically active and immunoreactive TNF-α were detected in LPS-stimulated blood from patients with PDR compared with patients with IDDM without retinopathy or healthy individuals. In contrast, significantly higher concentrations of both immunoreactive and biologically active TNF-α were observed in supernatants of LPS-activated mononuclear cells from patients with PDR compared with patients with IDDM without PDR or healthy subjects.

The present findings that low levels of both biologically active and immunoreactive TNF-α may be detected in total LPS-activated blood from PDR patients, but that isolated MNC from these individuals produce higher levels of TNF suggest that detection of high production of TNF-α by MNC in whole blood may be masked by the presence of increased serum levels of sTNF-R. This was later confirmed by observations that inactivation of sTNF-R by MoAbs caused an increase in the detection of biologically active TNF-α. These findings are supported by previous observations by others that high levels of sTNF-R interfere with the immunological detection of TNF-α [27] and that sTNF-R modulate the biological activity of TNF-α [28,29]. The mechanisms of control of TNF-α by TNF-R have been widely documented in vitro and in vivo [30,31], for which it is possible that high levels of sTNF-R present in the circulation of IDDM patients with PDR may play a crucial role in the control of TNF-α-mediated reactions at the retinal microvasculature. This is suggested by findings that sTNF-R spontaneously produced by rheumatoid tissue in culture exert a feedback control of TNF-α action [30], that injection of soluble TNF-RI prevents shock and mortality in mice as a result of LPS or TNF-α challenge [31], and that development of autoimmune diabetes mellitus in NOD mice may be prevented by transgenic expression of TNF-RI [32]. In addition, neuronal damage caused by ischaemic injury is exacerbated in mice lacking both TNF-R [33]. Although it is clear that sTNF-R may block TNF-α function, there is also evidence that they can prevent TNF-α inactivation and increase its half life in the circulation [34]. In this context, it may be also possible that sTNF-R might contribute to development of microvascular complications often observed in individuals with IDDM.

TNF-α induces changes in endothelial cell morphology and behaviour [34,35], promotes cytokine synthesis [1], mediates chemotaxis of monocytes and fibroblasts [36], induces synthesis of extracellular matrix proteins [37] and enhances the expression of vascular cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) [1]. Whilst these proinflammatory functions mediated by TNF-α are necessary for the development of neovascularization and fibroplasia in general, evidence that TNF-α is present in vitreous from eyes with PDR [17,18] and on infiltrating cells, vascular endothelium and extracellular matrix of PDR membranes [16], seriously implicates this cytokine in the pathogenesis of this sight-threatening complication of IDDM. This is further supported by observations that intravitreal injection of TNF-α in mice induces necrosis of endothelium and breakdown of the blood retinal barrier [38], and that TNF-α-deficient mice are resistant to developing lung fibrosis [39].

That susceptibility to IDDM is associated with both HLA and TNF-α gene polymorphism [6–10], and that these genes are located within the MHC region, suggest that impaired production and control of TNF-α may be strongly associated with the MHC-linked pathology observed in IDDM. Furthermore, in view of the close association of PDR with other microvascular abnormalities of IDDM such as nephropathy and neuropathy, it is conceivable that impaired TNF-α production may also be responsible for the development of these complications. Further studies may clarify any association between TNF-α and TNF-R production and general microvascular complications of IDDM. The mechanisms that trigger the production of TNF-α in IDDM are not known, but it is likely that local hypoxia induced by capillary occlusion may trigger TNF-α release, as observed with monocytic cell lines subjected to low oxygen levels in vitro [40]. It is also conceivable that high levels of protein modified by glucose metabolites such as methylglyoxal products, which are associated with development of microvacular complications in diabetes mellitus [41], may induce monocyte production of TNF-α production in vivo, as has been shown in vitro [42].

The present findings suggest that impaired mechanisms of TNF-α production and control may operate during the development of PDR, and may have important implications for the detection of susceptibility and progression of microvascular disorders in IDDM.

Acknowledgments

We are grateful to Dr D. L. Russell-Jones for allowing us to recruit patients attending the diabetic clinic at St Thomas'. We also thank The Trustees of the Gift of Thomas Pocklington for their invaluable support.

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