Tight junction proteins expression and modulation in immune cells and multiple sclerosis

Abstract

The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood-brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre-treatment levels and clinical response phenotype to interferon-β therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune-mediated diseases such as MS.

Fig 1

Expression of TJPs in different leukocyte subsets. Immunofluorescence staining of PBLs from healthy controls with antibodies against claudin 1 or claudin 5 (green) and CD20 (A,B), CD14 (C,D), CD3 (E,F), CD4 (G,H), CD8 (I,J), ZO-1 (K,L) and ZO-2 (M,N) (all red) and a nuclear dye (TO-PRO 3-blue). Scale bar: 5 μm. Representative results from three independent experiments are shown.

Fig 2

Claudin 1 is up-regulated in activated PBLs. PBLs from healthy volunteers were activated in vitro with either anti-CD3 antibody (α-CD3), PHA, PHA and IL2 or left without treatment (non-activated) and analysed by (A) immunofluorescence analysis with anti-claudin 1 antibodies (green) and a nuclear dye (TO-PRO 3 – blue). Scale bar: 5 μm. (B) Western blot analysis of claudin 1 protein levels. Bar graph depicts the fold change of claudin 1 protein levels relative to cells without treatment. The protein levels of claudin 1 were normalized to β-actin levels for each activation protocol using densitometry analysis. The data are presented as the mean ± S.E. of three independent experiments. *P = 0.04 (Student’s t-test). (C) Representative Western blot of claudin 1 expression in PBLs. PC: a positive control of extracts from HEK 293 cells transfected with a claudin 1 expression vector.

Fig 3

Expression levels of claudins 1 and 5 in leukocytes from MS and T1D. (A) Claudin 1 protein levels in PBLs of healthy controls (n = 13), OND controls (n = 14), T1D controls (n = 14), MS patients in remission (n = 13) and MS patients in relapse (n = 10). (B) The expression levels of CLDN5 RNA (2^ΔC_T) relative to reference genes were determined by real- time RT-PCR of PBL RNA derived from MS patients in relapse (n = 33), MS patients in remission (n = 39), and healthy controls (n = 18). (C) Claudin 5 protein levels in the PBLs of healthy controls (n = 13), OND controls (n = 14), T1D controls (n = 14), MS patients in remission (n = 11), and MS patients in relapse (n = 17). Box plot and data points display the relative protein levels determined by Western blot analysis. The relative intensity of claudin 1 or claudin 5 proteins compared to β-actin signals was determined by densitometry. Medians are depicted by horizontal bars.

Fig 4

GC treatment leads to a reduction in TJ expression in MS patients. TJ gene expression in PBLs from MS patients in relapse following therapeutic intravenous methylprednisolone GC treatment as determined by (A) real-time RT PCR analysis of paired samples from before and 7 days after GC treatment initiation for each donor. RNA relative levels described by 2^ΔΔC_T values for the genes JAM1, JAM3 and CLDN5 (n = 28 for each gene). (B) Bar graph depicting the claudin 5 protein levels before (pre) and following (post) GC therapy (n = 8). The relative intensity of claudin 5 compared to β-actin signals was quantified by densitometry using Western blot analysis; representative blots shown in bottom panel for three patients. PC: positive control of extract from HEK 293 cells transfected with a claudin 5 expression construct.

Fig 5

Pre-treatment CLDN5 mRNA levels are significantly lower in patients defined as ‘good responders’ to IFN-β treatment. The relative expression of CLDN5 RNA (2^ΔC_T) was determined by real-time RT-PCR in PBLs collected from patients defined as ‘good responders’ (n = 13) and ‘others’ –patients whose clinical response to IFN-β did not meet the criteria for ‘good responders’ (n = 9), before and after 3–6 months of IFN-β treatment.