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. 1999 Nov;118(2):213–218. doi: 10.1046/j.1365-2249.1999.01067.x

Platelet expression of tumour necrosis factor-alpha (TNF-α), TNF receptors and intercellular adhesion molecule-1 (ICAM-1) in patients with proliferative diabetic retinopathy

G A Limb *, L Webster *, H Soomro †, S Janikoun †, J Shilling †
PMCID: PMC1905412  PMID: 10540181

Abstract

Microvascular complications of insulin-dependent diabetes mellitus (IDDM) have been strongly associated with platelet abnormalities, whilst TNF-α has been implicated in the pathogenesis of this condition. However, at present it is not clear whether human circulating platelets express TNF-α or TNF receptors (TNF-R) or whether impaired expression of these molecules and of the TNF-reactive adhesion molecule ICAM-1 may be associated with platelet abnormalities in patients with IDDM. On this basis we investigated the platelet expression of these molecules in patients with IDDM complicated or uncomplicated by proliferative diabetic retinopathy (PDR) and in healthy subjects. We observed that the proportion of platelets staining for TNF-α was significantly higher in IDDM patients with active PDR than in patients without microvascular complications (P = 0.0078), quiescent PDR (P = 0.003) or healthy subjects (P = 0.0013). Patients with active PDR also showed a higher proportion of platelets expressing TNF-RI (P = 0.0052) and TNF-RII (P = 0.015) than healthy controls or patients with quiescent PDR (P = 0.009 and 0.0006, respectively). In addition, the percentage of ICAM-1+ platelets was significantly higher in patients with active PDR than in patients with quiescent PDR (P = 0.0065) or normal subjects (P = 0.013). There was a direct correlation between platelet expression of TNF-α and that of TNF-R in PDR patients, indicating that platelet staining for TNF-α may be due to binding of this cytokine to its receptors. The results suggest that increased platelet expression of TNF-α, TNF-R and ICAM-1 in IDDM patients may constitute important markers of thrombocyte abnormalities during the development of microvascular complications of diabetes mellitus.

Keywords: platelets, diabetic retinopathy, tumour necrosis factor-alpha, TNF receptors, intercellular adhesion molecule-1, insulin-dependent diabetes mellitus

INTRODUCTION

Proliferative diabetic retinopathy (PDR) is a common complication of insulin-dependent diabetes mellitus (IDDM), characterized by epiretinal outgrowth of new vessels and formation of neovascular membranes at the vitreoretinal interface [1]. Early histopathological features of PDR reveal capillary occlusion and retinal ischaemia [2], in which platelet adhesiveness and aggregation appear to play an important role [3,4]. Abnormalities in platelet size and function are also observed in patients with diabetes mellitus complicated by PDR [5], and although there is evidence that platelets produce cytokines responsible for cell extravasation, neovascularization and fibrosis [6,7], very little is known of the role that platelet-derived cytokines or interaction of platelet receptors with cytokines may play in this pathological complication of diabetes mellitus.

Several lines of research have implicated TNF-α in the pathogenesis of IDDM [8,9], and there are indications that genetic polymorphism for this cytokine play an important role in the development of PDR [10]. TNF-α is often present in vitreous [11,12] and in the luminal and abluminal surfaces of fibrovascular tissue from eyes with PDR [13], and individuals with this retinal complication exhibit abnormalities in the production of TNF-α and TNF receptors (TNF-R) when compared with disease and normal controls [14]. In addition, TNF-α induces platelets to release vasoactive molecules [15], promotes their adherence to vascular endothelium [16] and induces their expression of surface ICAM-1 [17]. This is of special relevance to the microvascular pathology observed in IDDM, as platelet ICAM-1 interaction with leucocyte LFA-1 molecules may cause cell aggregation, which together with increased platelet adherence to endothelium might constitute a crucial event in the development of capillary occlusion leading to microvascular complications [18].

The biological activity of TNF-α may be inhibited in vitro by TNF-R, known as TNF-RI (55 kD molecular weight) and TNF-RII (75 kD molecular weight) [19]. Although it has been shown that platelets bind TNF-α [20] and that murine megakaryocytes express TNF-RI [21], at present it is not clear whether normal human platelets express TNF-R, or whether these may be induced during pathological states such as microvascular complications of diabetes mellitus. On this basis, we investigated whether circulating platelets from IDDM patients with or without PDR, and those from normal individuals, stained for TNF-α, TNF-RI, TNF-RII and the TNF-α-reactive adhesion molecule ICAM-1. We also examined whether there was any relationship between platelet expression of TNF-α and that of TNF-R and ICAM-1.

PATIENTS AND METHODS

Ethical approval for this study was obtained from the St Thomas' Hospital ethical committee, and methods complied with the principles expressed in the Declaration of Helsinki. Forty-nine patients with IDDM attending the diabetic eye and medicine clinics were selected for the study upon prior written consent on the basis that their diabetes was of young onset (i.e. < 40 years of age), insulin-dependent, of at least 10 years duration, and that they either had developed PDR or that they had not presented with any form of retinopathy or other severe microvascular complications of IDDM. 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 and healthy controls included in the study

graphic file with name cei0118-0213-t1.jpg

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*Severe neuropathy.

†Minimal non-proliferative retinopathy.

‡Severe or quiescent proliferative retinopathy.

§Microalbuminuria.

¶Severe nephropathy.

Assessment of retinopathy

Diabetic individuals included in the study were divided into three main groups: (i) those with no retinopathy (n = 18), as judged by absence of microaneurisms, macular oedema or hard exudate formation by one 45° field fundus photography and direct ophthalmoscope; (ii) those with severe PDR (n = 17), as judged by new vessel proliferation, severe intraretinal vascular abnormalities, photocoagulation scars and preretinal or vitreous haemorrhages; and (iii) those with quiescent PDR (n = 13), 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 [22].

Platelet isolation from whole blood

Blood drawn without tourniquet (4.5 ml) was collected into 0.5 ml of 0.134 m EDTA containing 20 U/ml heparin. According to our published methods [23], the sample was immediately fixed for 10 min with an equal volume (5 ml) of 0.5% paraformaldehyde (PFA) in PBS to prevent platelet activation. Platelet-rich plasma was obtained by centrifugation at 350 g for 10 min. To avoid contamination with erythrocytes and leucocytes, only the upper two-thirds of the platelet-rich plasma were carefully removed by aspiration. Platelet-rich plasma was then diluted 1:5 with 3.8% sodium citrate in PBS and centrifuged at 1500 g for 15 min. Platelets obtained by this procedure were washed twice with 3.8% sodium citrate and finally resuspended in 100 μl of the same buffer. Platelet preparations were found to contain < 0.01% leucocytes as assessed microscopically.

Flow cytometry analysis of platelets

Platelet expression of TNF-RI, TNF-RII, ICAM-1 (CD54) and surface-bound TNF-α was determined by flow cytometry in a FACScan (Becton Dickinson, Mountain View, CA). Using polystyrene tubes (Becton Dickinson, NJ), platelets (10 μl) were incubated on ice for 30 min with equal volumes of optimal dilutions of MoAb MAS485 (Harlan, Sera Lab, Loughborough, UK) and MoAb B-C7 (Serotec, Oxford, UK) detecting TNF-α, MoAb MR1-2 detecting TNF-RI, MoAb MR2-1 detecting TNF-RII (HyCult Biotechnology; Bradsure Biologicals Ltd, Loughborough, UK), MoAb BBIG-I1 detecting ICAM-1 (R&D Systems, Abdingdon, UK), or MoAb PM6/248 detecting the platelet marker CD41 (Serotec). After incubation platelets were washed twice with 3.8% sodium citrate buffer. FITC-labelled F(ab)2 fragments of rabbit anti-mouse antibody (25 μl) (Dakopatts, Glostrup, Denmark) were then added and the platelets were incubated for a further 30 min on ice. Finally, platelets were washed twice with cold citrate buffer and resuspended in 300 μl of 0.5% PFA in PBS for FACS analysis. Non-specific background fluorescence was determined by using mouse IgG1 isotype antibody (Serotec), as well as a FITC-labelled F(ab)2 preparation of rabbit anti-mouse immunoglobulins (Dako, Glostrup, UK). Using forward and side scatter dot plots, gates were set on platelets with exclusion of erythrocytes and leucocytes. The purity of cells within this gate was assessed using antibodies to the CD41 molecule, which specifically stains megakaryocytes and platelets. To confirm that the staining for TNF-α, TNF-R and ICAM-1 was associated to platelets, we performed dual staining for CD41 and TNF-α or CD41 and TNF-R in several samples using CD41 antibody conjugated with RPE (Dako).

Statistical analysis

The significance of difference between corresponding groups of observations was evaluated by the Mann–Whitney U-test. Pearson's correlation coefficient was used to determine the relationship between the proportion of platelets expressing TNF-α and that of platelets expressing TNF-RI, TNF-RII and surface ICAM-1. Acceptable significance was recorded when P < 0.05.

RESULTS

Staining for TNF-α by platelets from patients with IDDM complicated by PDR; comparison with platelets from disease and healthy controls

As shown in Fig. 1, the proportion of platelets staining for TNF-α was significantly increased in IDDM patients with active PDR (range 0.32–14.5%) when compared with patients with quiescent PDR (range 0.1–1.9%; Mann–Whitney U-test, P = 0.000 36), IDDM patients without retinopathy (range 0.41–4.23%; Mann–Whitney U-test, P = 0.0078) or healthy individuals (range 0.35–1.86%; Mann–Whitney U-test, P = 0.0013). There were no differences in the levels of TNF-α-positive platelets between individuals with quiescent PDR and IDDM patients without retinopathy or healthy controls (P > 0.05).

Fig. 1.

Fig. 1

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Proportion of platelets staining for TNF-α in individuals with insulin-dependent diabetes mellitus (IDDM) complicated or uncomplicated by proliferative diabetic retinopathy (PDR), quiescent PDR and healthy subjects. Mann–Whitney U-tests: *P = 0.000 36 (versus quiescent PDR), P = 0.0078 (versus IDDM without PDR), P = 0.0013 (versus healthy controls). The lines represent the median of the percentage of positive platelets in each group.

Staining for TNF-RI and TNF-RII by platelets from IDDM patients complicated by PDR; comparison with platelets from disease and healthy controls

Figure 2 shows that the proportion of platelets staining for TNF-RI was significantly higher in IDDM patients with active PDR (range 0.43–37.8%) than in patients with quiescent PDR (range 0.2–3.87%; Mann–Whitney U-test, P = 0.009) or healthy controls (range 0.001–3.1%; Mann–Whitney U-test, P = 0.0052). Levels of TNF-RI- positive platelets were also higher in patients with IDDM without retinopathy (range 0.3–6.2%) than in healthy subjects (Mann–Whitney U-test, P = 0.045). However, no differences were observed between IDDM patients with active or quiescent PDR and patients with IDDM without retinopathy (P > 0.05).

Fig. 2.

Fig. 2

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Proportion of TNF-R-positive platelets in patients with insulin-dependent diabetes mellitus (IDDM) complicated or uncomplicated by proliferative diabetic retinopathy (PDR) and healthy controls. Mann–Whitney U-tests: *P = 0.009 (versus quiescent PDR), P = 0.005 (versus healthy controls); **P = 0.045 (versus healthy controls); ***P = 0.0006 (versus quiescent PDR), P = 0.015 (versus healthy controls); ****P = 0.004 (versus quiescent PDR). The dotted line represents the median of the number of TNF-RI- or TNF-RII-positive platelets in healthy subjects.

The proportion of platelets staining for TNF-RII was also higher in IDDM patients with active PDR (range 0.6–13%) than in patients with quiescent PDR (range 0.23–1.43%; Mann– Whitney U-test, P = 0.0006) or healthy controls (range 0.23–2.9%; Mann–Whitney U-test, P = 0.015). IDDM patients without retinopathy showed a higher proportion of TNF-RII-positive platelets (range 0.06–5.1%) than patients with quiescent PDR (Mann–Whitney U-test, P = 0.004). There were no significant differences in the levels of TNF-RII-positive platelets between individuals with IDDM alone and healthy subjects.

Staining for surface ICAM-1 by platelets from IDDM patients complicated by PDR; comparison with platelets from disease and healthy individuals

Figure 3 shows that the proportion of platelets staining for ICAM-1 was significantly higher in IDDM patients with active PDR (range 0.78–19.1%) when compared with patients with quiescent PDR (range 0.23–3.03%; Mann–Whitney U-test, P = 0.0065) or healthy individuals (range 0.32–2.13%; Mann–Whitney U-test, P = 0.013). Similarly, patients with IDDM without retinopathy exhibited higher numbers of ICAM-1-positive platelets (range 0.66–6.98%) than IDDM patients with quiescent PDR (Mann–Whitney U-test, P = 0.036) or normal individuals (Mann–Whitney U-test, P = 0.047). No difference in the proportion of platelets staining for ICAM-1 was observed between individuals with IDDM complicated by PDR and those without retinopathy (P > 0.05).

Fig. 3.

Fig. 3

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Percentage of platelets staining for ICAM-1 in patients with insulin-dependent diabetes mellitus (IDDM) complicated or uncomplicated by proliferative diabetic retinopathy (PDR), quiescent PDR and healthy subjects. Mann–Whitney U-tests: *P = 0.0065 (versus quiescent PDR), P = 0.013 (versus healthy subjects); **P = 0.036 (versus quiescent PDR); P = 0.047 (versus healthy subjects). The dotted line represents the median of the number of ICAM-1-positive platelets in healthy subjects.

Relationship between platelet expression of TNF-R and staining for TNF-α in IDDM patients with active PDR

As shown in Fig. 4, there was a direct correlation between the proportion of platelets staining for TNF-α and that of platelets expressing TNF-RI (Pearson's correlation coefficient; r = 0.92, P < 5.13−9) or TNF-RII (Pearson's correlation coefficient; r = 0.74, P = 0.000 17). There was no relationship between platelet expression of TNF-α and that of surface ICAM-1 (Pearson's correlation coefficient; r = 0.1, P = 0.65) (Fig. 4) or the expression of TNF-R and that of ICAM-1 (Pearson's correlation coefficient; P > 0.5, not shown).

Fig. 4.

Fig. 4

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Relationship between the expression of TNF-α and that of TNF-RI, TNF-RII and ICAM-1 in platelets from individuals with insulin-dependent diabetes mellitus (IDDM) complicated by proliferative diabetic retinopathy (PDR).

DISCUSSION

The key finding of this study was the observation that in IDDM patients with active PDR there is a significant increase in the percentage of platelets staining for TNF-α, when compared with healthy subjects or IDDM patients with quiescent PDR or without microvascular complications. The proportion of platelets expressing TNF-RI, TNF-RII and ICAM-1 was also higher in patients with active PDR than in patients with quiescent PDR or healthy subjects.

The significance of TNF-α binding to platelets lies in that this cytokine induces thrombocyte processes known to contribute to the microvascular pathology observed in PDR, such as platelet aggregation and adhesion to endothelium [3,4], and production of vasoactive agents, such as nitric oxide, free radicals and prostacyclin [24]. Since platelets and megakaryocytes are known to secrete TNF-α [6,7,25], platelet staining for this cytokine may be due to its cytoplasmic presence. However, the findings that platelet staining for TNF-α directly correlates with the expression of TNF-RI and TNF-RII may also indicate that TNF-α detected on the platelet surface may be bound to its receptors. Although the antibodies used in the present study block soluble TNF-α binding [14], it is possible that we are detecting bound cytokine, as the antibodies could recognize peptide sequences adjacent to the TNF-α binding epitope on the platelet membrane.

The observations that the proportion of platelets staining for TNF-α and its receptors is lower in patients with quiescent PDR than in patients with active PDR suggest that pan-retinal photocoagulation may cause a reduction in the local production of factors responsible for platelet activation. This is supported by recent findings, that laser photocoagulation induces changes in growth factor expression in the pig retina [26], and that production of transforming growth factor-beta 2 (TGF-β2) by retinal pigment epithelial cells is increased by laser photocoagulation [27]. Although platelets from patients with quiescent retinopathy were not examined before and at various time intervals after photocoagulation, the present findings merit further investigation to examine the effect of laser treatment on the expression of platelet activation markers.

Diabetic retinopathy is often accompanied by other microvascular complications, such as nephropathy and neuropathy [1]. In this context, it is possible that platelet expression of TNF-α, TNF-R and ICAM-1 may not constitute a specific marker of PDR but a marker of diabetic microangiopathy. We did not observe differences in the expression of these molecules between patients with PDR alone and patients with PDR accompanied by nephropathy (data not shown), possibly due to the small number of cases investigated. However, a large follow-up study may elucidate the implications of the present findings, and may clarify whether platelet expression of these molecules precedes or follows the manifestation of microvascular pathology in IDDM.

Platelet binding of TNF-α has been previously shown [20] and expression of TNF-RI by murine megakaryocytes was recently reported [21]. Although it has been suggested that platelet activation by TNF-α may be mediated through TNF-R [28], evidence for the expression of TNF-R on human platelets has not yet been documented. Our observations that platelets express TNF-R is in accordance with the demonstration of functional receptors for the cytokine interferon-gamma (IFN-γ) [29], and the observations that platelets from patients with Crohn's disease and ulcerative colitis show an enhanced expression of receptors for IL-1, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) [30].

Studies of rat models of diabetes mellitus show that platelet aggregation and microthrombus formation occur during retinal vessel occlusion leading to diabetic retinopathy [31], thus resembling those features observed in patients with diabetic retinopathy [3]. It is therefore possible that bound TNF-α may cause platelet sequestration in microvessels, by the same mechanism proposed to cause sequestration of leucocytes with membrane-bound TNF-α [32]. This is of special relevance to PDR, as TNF-α has been implicated in the pathogenesis of IDDM and its microvascular complications. High serum levels of TNF-α precede and accompany the onset of IDDM [8], cells expressing mRNA coding for TNF-α are found in the islets of Langerhans during early stages of the disease [9], injection of TNF-α into female newborn non-obese diabetic (NOD) mice accelerates the development of diabetes mellitus [33], and patients with IDDM complicated by PDR produce increased levels of TNF-α and TNF-R [14]. In addition, TNF-α is found in the extracellular matrix, endothelium and vessel walls of epiretinal membranes of PDR [13], and it is often present in vitreous from eyes with this complication [11,12].

TNF-α induces the expression of vascular cell adhesion glycoproteins on a wide number of cells [34], and up-regulation of ICAM-1 on human endothelial cells has been shown to be dependent upon binding of TNF-α to TNF-RI [35]. However, in experimental cerebral malaria, whose microvascular pathological features resemble those of PDR, it is the binding of TNF-α to TNF-RII that causes up-regulation of ICAM-1 on the platelet surface [36]. Hence, from the present findings we suggest that enhanced platelet expression of surface ICAM-1 in IDDM patients may be caused by TNF-α binding to platelet receptors. It is of interest that the proportion of platelets expressing TNF-α, TNF-R and ICAM-1 was similar to that reported for markers of platelet activation during diabetic microangiopathy, such as CD62P and CD63 [37,38]. This suggests that increased expression of TNF-α and related molecules may also constitute a marker of platelet activation during microvascular complications of diabetes mellitus.

From the present results we conclude that enhanced platelet expression of TNF-R, TNF-α and ICAM-1 in patients with IDDM complicated by PDR may be associated with the abnormal TNF-α production observed in these patients [14]. Moreover, platelet binding of TNF-α may contribute to the amplification of fibrovascular and fibrocellular functions mediated by this cytokine and which may be responsible for the microvascular complications of IDDM. Elucidation of the mechanisms that control TNF-R expression and TNF-α binding by platelets may have important implications for the treatment and prevention of proliferative diabetic retinopathy.

Acknowledgments

The authors especially thank Dr D. L. Russell-Jones for allowing them to recruit patients attending his diabetic clinic at St Thomas' Hospital. They also thank The Henry Smith Charity for its invaluable support.

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