Enzymatic cleavage of chemokines CCL2 and CXCL10 : Implications for multiple sclerosis pathogenesis.

In multiple sclerosis (MS), lymphocytes and monocytes penetrate the blood brain barrier (BBB), causing inflammation and leading to myelin damage and subsequent loss in the white matter of the central nervous system (CNS). Chemotactic cytokines (chemokines), contribute to the recruitment of immune cells into the CNS, and are expressed by astrocytes, the most abundant cell type in the CNS, and endothelial cells. CCL2 and CXCL10 are chemokines expressed in MS lesions, which are thought to be pivotal in chemoattraction of T cells and monocytes into the CNS. Astrocytes, microglia, and endothelial cells produce proteases, such as matrix metalloproteinases (MMPs), which contribute to the destruction of the BBB, facilitating cellular entry. Increased expression of MMP2 and MMP9, amongst others, has been shown previously in autopsied MS brain, and increased levels of the cell surface peptidase CD26, expressed by T cells and endothelial cells, have been linked to disease activity. Interactions between proteases and chemokines expressed in the same milieu can result in chemokine processing that dramatically increases or decreases their activity. To assess the potential effects of chemokine cleavage in MS, recombinant proteins were used in an in vitro study which examined processing of CCL2 and CXCL10 by MMP2, MMP9 and CD26, using mass spectrometry to identify enzymatic cleavage products. MMP9 removed four residues, and CD26 cleaved a dipeptide, from the N-termini of both CCL2 and CXCL10. MMP2 N-terminally cleaved four residues from CCL2, and five from CXCL10. C-terminal truncation of CXCL10 was observed with MMPs 2 and 9, which each removed four residues.Cleavage by CD26 was rapid, and complete with CXCL10 within 30min. MMP truncation of CCL2 was complete within 3h, but with CXCL10, remained incomplete by 48h. Astrocyte supernatant was examined by mass spectrometry for the presence of these truncated chemokines, but low levels prevented detection. Protease expression was investigated, to identify the likelihood that cleaved CCL2 and CXCL10 arise in vivo in MS. The MMP mRNA expression profile of cytokine-treated and untreated astrocytes examined by real time PCR showed that MMP2 mRNA was highly expressed with and without cytokine treatment, and MMP9 mRNA was increased following treatment with the pro-inflammatory cytokines tumour necrosis factor and interleukin-1 p. MS brain tissue was examined using dual-labelling immunofluorescence for CD26 expression, which was found in lesions to be associated with T cells and monocytes in perivascular cuffs, and macrophages within the parenchyma, but not with astrocytes. The chemotactic activity of the cleaved forms of CCL2 and CXCL10 identified in this study was investigated using real time in vitro migration assays. THP-1 cells, a monocytic cell line, exhibited a two-fold reduction in migration to cleaved CCL2 isoforms, compared to the intact form. Preliminary experiments with Jurkat cells, a T cell line, indicated that migration remained unaffected, or was slightly increased, by cleaved CXCL10 compared to the intact form. Collectively, this study demonstrated that cleavage of CCL2 and CXCL10 by proteases, found at elevated levels in MS, may be an important regulator of chemokine activity. In particular, CD26 may play a key role in regulating chemokine activity, and thus cell migration into the CNS, as it demonstrated rapid and highly specific proteolytic activity and is highly likely to encounter chemokines as it was associated with T cells and macrophages that express the appropriate chemokine receptors.