In this study we aimed to explore what proteins are expressed in the inflammatory foci of CLE and DM lesions. Our major finding from tandem mass spectrometry investigation is that IL-16 is the only detectable and highly abundant cytokine in CLE, but not DM lesions.
The study is unique in several aspects. We utilized a novel method – laser capture microdissection which allowed excision of the cells comprising inflammatory foci. The collected tissue was analyzed in a hypothesis-free manner using mass-spectrometry technique. The detected proteins were compared among the groups CLE, DM and HC. Data analysis using string-db.org database revealed the involved protein networks, similarities, and also some major differences between the conditions.
Increased levels of circulating IL-16 in SLE patients has been described before, by us and others[16, 17]. Also, upregulation of IL-16 has been observed in psoriasis, systemic sclerosis, inflammatory bowel disease and several malignancies[17–19]. Our group has previously reported observation that SLE patients with active nephritis or arthritis, had high levels of circulating IL-16, while patients with CLE had lower[17].The presented data indicate, that cytokines in the circulation does not necessarily correspond the molecular processes taking place in the LE target organ, for example skin.
Interestingly, other investigators described that in the skin affected by systemic sclerosis, approximately 1/3 of the infiltrating cells express IL-16 [18]. In comparison, in the current study we found that approximately 2/3 of the infiltrating cells in CLE carry this molecule, while proportion in DM, PC and HC was about 1/3, similar to that described systemic sclerosis[18]. The predominant sources of IL-16 are T cells, but also eosinophils, DCs, mast cells, macrophages and monocytes can also produce IL-16[12, 13]. IL-16 is generated as a precursor molecule, and when cleaved by caspase-3, two molecules with different functions are generated: N-terminal pro-IL-16 and C-terminal secreted/mature IL-16. Pro-IL-16 molecule functions as a regulator of T cell growth, and a secreted mature IL-16 functions as a CD4 and/or CD9 ligand and facilitate cell motility and activation[12, 13]. It is known, that majority of infiltrating cells in the skin are T-cells, and as our results indicate, they carry the IL-16 molecule. We suggest that IL-16 could function as a chemoattractant in the CLE lesions. Also, intranuclear expression of IL-16 is known to impede cell cycle progression and could possibly negatively affect cell growth and regeneration within the skin lesions[19].
Multiple cytokines have been found to be involved and mediate local inflammatory responses in CLE including type I and III IFNs, TNF-α, IL-1β, HMBG1, as well as the Th17 pathway along with IL-21[14–17]. Our analysis confirmed that IFN regulated protein (IRP) network is the most abundant protein pathway activated in the lesions of both CLE and DM. While this prominence of the IRP network was similar between the two diseases, CLE lesions appeared to have higher expression and diversity of the proteins within IFN-pathway.
The transporters associated with antigen processing (TAP-1 and TAP-2) are of importance for normal expression of MHC class I and presentation of intracellular peptides, while TAPBP is a catalyst molecule in the binding of antigen. Defective function of this pathway is implicated in type I autoimmune diabetes mellitus[20]. High expression of these molecules in both CLE and DM suggest that autoantigen presentation could be occurring intracutaneously in inflammatory infiltrates and could be of interest in further investigation.
Aminoacyl-tRNA synthetases (ARS) is another abundant molecular group detected in the lesions of both CLE and DM, but not in the control skin. The detected ARS included tryptophanyl (WARS) and threonyl (TARS) as the most abundant. WARS has been demonstrated to have additional functions in the immune system, including regulation of IFN-γ production and this could be of importance in both CLE and DM[21].
Detection of complement components (C1q, C3 and C4) and immunoglobulins (Ig) at the dermo-epidermal junction (the so-called lupus band) has been used in CLE diagnostics in many decades using direct immunofluorescence technique[22]. The test has been criticized for its limited specificity and sensitivity[23]. Deposition of MAC within the CLE and lupus nephritis inflammatory infiltrates has been reported earlier [24, 25]. Also, detection of MAC within the endothelium and perifascicular, usually atrophic, muscle fibers is utilized as a distinct diagnostic feature for DM [10]. Proteomic findings indicate that activation and deposition of complement, including membrane attack complex (MAC) is more abundant in CLE cutaneous inflammatory foci, than DM, where intramuscular MAC activation seems to play more important role[10].
The limitations of this study include limited amount of individuals included per each group (5 cases per group) that were run in the proteomic analysis, however the actual findings where confirmed in higher number of cases (CLE (n = 13) and DM (n = 7), and both PC (n = 5) and HC (n = 5)). We acknowledge that less stable proteins could possibly been destroyed during the biopsy handling and those lower abundant could been missed by mass-spectrometry analysis, since IL-16 was the only cytokine that was abundantly detected by this technique.
In conclusion, using a novel technique laser capture microdissection combined with hypothesis free mass-spectrometry investigation we identified that IL-16 is abundant and the only detectable cytokine in inflammatory foci of CLE lesions. We confirmed high expression of IL-16 by IHC, and also detected focal expression of caspase-3, the enzyme that cleaves IL-16 into its active forms. Abundant deposition of components of classical complement activation pathway is another feature of CLE, while abundant expression of IFN regulated proteins is a characteristic of both CLE and DM. Our findings could be useful in diagnosing and differentiating CLE and DM in difficult cases, if validated clinically. These observations offer novel information on molecules involved in disease mechanisms and propose a novel pathway to be explored in search for CLE therapeutic targets.