We investigated the ability of SLE patients and control individuals to activate latent TGF-β1
in an in vitro
assay that utilizes the physiological activation of latent TGF-β1
that occurs normally during blood clotting. The activation of TGF-β1
during clotting is complex, being mediated through several mechanisms [19
] involving protease (plasmin) activation and interaction of TGF-β1
with thrombospondin-1. Using an ELISA assay validated for detection of active TGF-β1
], we determined the increased active TGF-β1
after clotting a standard volume of blood at 37°C for 90 minutes relative to the nonclotted sample. Although no differences in mean values were observed between AIs of control individuals and SLE patients, we hypothesized that the level of biological variation in the SLE group could be used as a surrogate marker of the efficiency of activating latent TGF-β1
. This would allow us to establish whether low or high TGF-β1
activation efficiency could be linked with known abnormalities in lymphocyte apoptosis and markers of early atherosclerosis.
In accordance with other studies [21
], we found an increase in apoptosis in the PBMCs of SLE patients compared with control individuals following 24 hours in culture. We found a lower rate of apoptosis at 24 hours (median 3.35%) compared with that described by Emlen and coworkers [21
] (mean 12%). However, our SLE patients have a low disease activity score (mean SLEDAI score 1.75) and low damage score (mean SLICC score 1.1). This is consistent with the finding reported by Emlen and coworkers of a significant positive correlation between disease severity (SLAM [Systemic Lupus Activity Measure] index) and rate of apoptosis.
There was no significant difference after 24 hours of culture between the levels of apoptosis in patients receiving and those not receiving steroids at the time of study. In both patients and control individuals we observed a significant inverse relationship between level of PBMC apoptosis and TGF-β1 activation index (low TGF-β1 AI linked with high level of apoptosis).
The significance of increased PBMC apoptosis in SLE is profound, possibly reflecting increased levels of cells undergoing activated induced cell death and/or a defect in non-inflammatory phagocytosis of apoptotic cells. Failure to achieve programmed cell death and to clear apoptotic cell fragments could be a key pathogenic factor in the development of autoimmunity. As demonstrated in TGF-β1
knockout mouse, a loss of control in apoptosis affects the development and control of tolerance. Lack of TGF-β1
leads to increases in the levels of both the number of activated T cells and the levels of apoptosis in activated T cells and self-tolerant T cells – a situation that may be similar to that found in SLE patients. In the present study we report, for the first time, a significant association between ability to activate TGF-β1
and the degree of PBMC apoptosis at 24 hours. In the TGF-β1
knockout mouse there is an increase in mitochondrial membrane potential, and such increases are associated with initiation of apoptosis. It was recently demonstrated that the mitochondrial membrane potential in SLE patients is also increased [24
]. Those SLE patients with low TGF-β1
AI status/increased apoptosis may be most at risk for the fundamental inflammatory process that drives SLE autoantibody production.
It is now well established that SLE patients are at fivefold to ten-fold increased risk for coronary heart disease compared with the general population. Classic risk factors have been found to be of importance in promoting the development of atherosclerosis in SLE [6
]. However, after adjusting for Framingham risk factors, a significant excess risk remains [25
]. This suggests that additional factors contribute to atherogenesis in SLE. Additional factors at play in SLE may include other metabolic changes such as renal impairment and homocysteine as well as adverse effects of steroid therapy and factors related to the underlying disease process, such as endothelial dysfunction and immune complex deposition [26
The inverse correlation of TGF-β1
activation status and LDL-cholesterol levels identified in patients but not in control individuals is therefore highly relevant to this inflammatory process in the vascular wall. Two potential mechanisms whereby LDL might reduce TGF-β1
function have been described. First, it has been shown that very-low-density lipoprotein and LDL can inhibit the binding of active TGF-β1
to the type II TGF-β receptor and thereby suppress signalling through the receptor [27
]. Second, and with particular relevance to this study, oxidized LDL is reported to interact specifically with thrombospondin-1 and inhibit the thrombospondin-1 dependent activation of latent TGF-β1
]. In SLE, Nuttal and coworkers [29
] noted that LDL-cholesterol was more likely to exist as small dense particles that are more prone to oxidation. Although we did not measure LDL particle size, this difference in the type of LDL present in patients and control individuals may explain our observation of an inverse correlation of TGF-β1
AI and LDL in SLE patients, which was not seen in control individuals.
In the present study carotid IMT itself was not different between patients and control individuals; this is consistent with the findings of other larger series of SLE patients. Indeed, Roman and coworkers [30
] found lower carotid IMT in SLE. Low TGF-β1
activation was also strongly associated with increased carotid IMT, an early marker of atherosclerotic change. It has been proposed that low levels of active TGF-β1
in the artery wall, resulting from apolipoprotein(a) inhibition of plasminogen activation and failure to activate latent TGF-β1
through plasmin proteolysis, allows endothelial and smooth muscle cell proliferation, leading to intima-medial expansion [8
]. In our study this relationship was observed only in SLE patients, and the slopes in the SLE and control groups were significantly different (P
= 0.0001), suggesting that the TGF-β1
interaction with IMT in SLE patients is different from that in age-matched/sex-matched control individuals. Although the TGF-β1
AI did not differ significantly between patients and control individuals, in the context of SLE increased oxidized LDL may promote a low TGF-β1
milieu, permitting excessive cellular apoptosis and enhancing the propensity for atherogenesis. Further studies are now needed to explore this hypothesis and it may be that several different factors govern the progression of carotid IMT in SLE. Such prospective studies will be needed to explore the interaction between inflammation and early atherosclerosis in more detail both in patients and unaffected individuals.
The association of low TGF-β1 AI and disease duration suggests that prospective studies of patients might identify changes in TGF-β1 activation and lipoprotein subfractions over time that could influence the development of atherosclerosis in SLE. Some of the atherogenic risk associated with LDL, in particular oxidized LDL, could be mediated through modulation of the availability of active TGF-β1 in the vasculature.
This observation has wider applications for prospective monitoring of TGF-β1 activation, not only in SLE but generally in patients developing atherosclerosis and fibrosis (for example, chronic allograft rejection). Therapeutic manipulation of the levels of active TGF-β1 may offer a new perspective in controlling the expression of disease in patients with SLE.