We demonstrate that the signaling potential of LIGHT is affected by two polymorphisms at positions E214K and S32L. The 214 position in LIGHT directly influenced the binding avidity to a neutralizing human antibody and the LTβR, indicating this residue is located in or near the receptor-binding region. This result provides an important consideration for the use of inhibitors of LIGHT, such as decoy receptors or antibody, in analyzing patient responses in clinical trials. The 32L variant located in the intracellular domain lowered the avidity of binding to DcR3 and decreased the membrane expression of LIGHT. Increased bioavailability of LIGHT due to lower avidity for DcR3, and enhanced binding avidity of LIGHT for LTβR could combine to increase signaling activity and predispose to unwarranted inflammation.
The transcriptional regulation of LIGHT
, like TNF
, may also contribute to its bioavailability. Several polymorphisms located in the 5' promoter region impact the transcriptional activity of LIGHT
). Studies in mice indicated that sustained expression of LIGHT in T cells caused significant pathology (17
). Interestingly, the promoter haplotype polymorphism reported by Kong et al. (39
) showed the haplotype with low expression was associated with vascular dementia. Although the opposite of what might have been predicted, this result implies a role for shedding and soluble LIGHT, perhaps acting through HVEM-BTLA pathway to protect the brain vasculature. This notion is supported by the experiments that demonstrated the HVEM-BTLA pathway protects the intestinal mucosa from immune damage (40
LIGHT is also controlled by cleavage from the cell surface and binding to soluble decoy receptors. LIGHT is shed from the cell surface by proteolysis creating a soluble form that can bind all three of its receptors (29
). Our data showed that EL4 cells expressing the LIGHT32L variant shed a reduced level of soluble LIGHT relative to the reference 32S. Although it is reasonable to deduce that the S32L substitution might have an negative impact on the shedding of LIGHT, the reduction of shed LIGHT from the 32L variant may well reflect on the amount of LIGHT present on the cell surface.
In addition, the LIGHT variant 32L-214E and 32S-214K in heteromeric complexes decreased the avidity for DcR3 and limited the capacity of DcR3 to inhibit activation of the LTβR by cell associated LIGHT. The mechanism of how this combination of LIGHT variants in the heterotrimers altered DcR3 avidity is not clear. It could be a direct effect on binding, or an indirect effect, perhaps related to the stability of LIGHT trimer, or modification of this putative phosphorylation site provides another intriguing possibility. Studies in progress are addressing this issue. However, the distinctive binding characteristics of the LIGHT variants in relation to DcR3 and LTβR are likely to have an impact on the bioavailability of both LIGHT and DcR3 in the tissue microenvironment, which may influence immune activation and inflammation.
Patients with active rheumatoid arthritis (21
) have elevated serum levels of soluble LIGHT, implicating the potential contribution of LIGHT to the autoimmune disease process. Concurrently, elevated levels of DcR3 were identified in the synovial fluid and serum, and in tissues with active disease from patients with RA and SLE, but not osteoarthritis, implicating the DcR3 is a component of the inflammatory response mechanism. Our results are supported by studies from Bamias et. al., (41
) and Hayashi et. al., (42
) that showed increased DcR3 in RA and other reports of elevated DcR3 in SLE (43
). In RA patients, the constitutive expression of DcR3 mRNA was found to be at the similar level in normal and disease tissue, indicating that expression of DcR3 protein is probably under dynamic control by post-translational processes. Moreover, the bioavailability of free DcR3 in the synovial fluid and serum is likely to be influenced by the presence of other DcR3 binding molecules, such as TL1A, FasL, as well as heparin.
Our data showed that the LIGHT polymorphisms altered binding avidity to DcR3. Although an elevated level of DcR3 was strongly associated with active RA, the physiological role of DcR3 in the pathogenesis of autoimmune RA remains to be further defined. We suggest the possibility that DcR3 could play an enhancing role in autoimmune-mediated responses by blocking soluble LIGHT binding the HVEM-BTLA cis
complex as an inhibitory signaling mechanism (36
). This hypothesis is supported by the development of an autoimmune-like syndrome in mice expressing human DcR3 transgene, which lack the DcR3 gene, potentially revealing a role for LIGHT, TL1A or FasL (45
In addition, LIGHT, LTβR and DcR3 may play a role in liver inflammation (17
) and the development of hepatocellular carcinoma (HCC) (46
). Chronic liver inflammation associated with Hepatitis B and C virus infections may be a contributing factor for the development of HCC. Two recent studies showed elevated DcR3 levels in the clinical isolated HCC samples (48
). Furthermore, Haybaeck et al. recently demonstrated a functional link between LTβR signaling and the formation HCC (50
). Intriguingly, relative to Caucasians, there is a significantly higher incidence liver cancer in the African American and Asian groups (51
), which also have higher allelic frequencies of the LIGHT-214K polymorphism. Our current findings, in particular, the distinctive binding features of the LIGHT variants in relation to DcR3 and LTβR, suggest the LIGHT polymorphisms could play a pathogenic role in the development and progression of liver inflammation and the formation of hepatocellular carcinoma.
Our results provide mechanistic insight into the effect of the polymorphic variants on signaling and bioavailability of LIGHT and DcR3. These LIGHT variants, acting individually or in concert as a haplotype, can alter the signaling potential of LIGHT. Variants in other components of the LIGHT cosignaling circuit, such as HVEM, BTLA, CD160 or LTβR might create haplotypes with disease linkages, although at this time such haplotypes are unknown. Pathogens place powerful selective pressures on genes involved in controlling immune responses. The evidence that α- and β-herpesvirus specifically target pathways involving LIGHT (7
) provides an argument for the natural selection of variants that might be more effective in controlling virus infections. A consequence of this natural selection of enhanced signaling may predispose towards the development of autoimmune disease and/or cancer, the cut of the proverbial double-edged sword.