Expression of RhoA by inflammatory macrophages and T cells in rat experimental autoimmune neuritis

Abstract

RhoA is one of the best-studied members of Rho GTPases. Experimental autoimmune neuritis (EAN), which is characterized by infiltration of T cells and macrophages into the peripheral nervous system, is an autoantigen-specific T-cell-mediated animal model of human Guillain-Barré Syndrome. In this study, RhoA expression has been investigated in the dorsal/ventral roots of EAN rats by immunohistochemistry. A significant accumulation of RhoA+ cells was observed on Day 12, with a maximum around Day 15, correlating to the clinical severity of EAN. In dorsal/ventral roots of EAN, RhoA+ cells were seen in perivascular areas but also in the parenchyma. Furthermore, double-labelling experiments showed that the major cellular sources of RhoA were reactive macrophages and T cells. In conclusion, this is the first demonstration of the presence of RhoA in the dorsal/ventral roots of EAN. The time courses and cellular sources of RhoA together with the functions of RhoA indicate that RhoA may function to facilitate macrophage and T-cell infiltration in EAN and therefore could be a potential therapeutic target.

1

The time course of RhoA⁺ cell accumulation in dorsal and ventral roots of EAN rats. (A) Clinical score of EAN severity. Rats (n = 6) were immunized with synthetic neuritogenic P2 peptide and monitored for development of EAN. Severity of disease was graded as follows: 0, normal; 1, reduced tonus of tail; 2, limp tail, impaired righting; 3, absent righting; 4, gait ataxia; 5, mild paralysis of the hind limbs; 6, moderate paraparesis; 7, severe paraparesis or paraplegia of the hind limbs; 8, tetraparesis; 9, moribund; 10, death. Results are given as mean clinical score ± SEM. (B) The time course of lesional RhoA⁺ cell accumulation in dorsal and ventral roots of EAN rats. After immunostaining, the whole section was scanned at 40 times magnification for the ventral or dorsal roots of highest RhoA⁺ cell accumulations (hot spots), followed by counting the number of RhoA⁺ cells within a single high-power field (HPF, x400 magnification) in each hot spot. Five hot spots were counted for each section and the mean value was taken. In each field studied, only positive cells with the nucleus at the focal plane were counted. Results were given as arithmetic means of positive cells per HPF and SEM. Statistical analysis was performed by one-way ANOVA followed by Dunnett's Multiple Comparison test (Graph Pad Prism 4.0 software).

2

Immunohistochemical labelling of RhoA in spinal cord dorsal and ventral roots of normal and EAN rats. (A) Immunostaining of spinal cord dorsal and ventral roots without the primary antibody as a negative control. (B) RhoA expression was absent in dorsal/ventral roots of normal adult rats. (C) Accumulation of RhoA⁺ cells was seen in dorsal/ventral roots Day 15 after immunization in EAN. RhoA⁺ cells accumulated near blood vessels (D), but were also seen in the parenchyma (E). (F–I) RhoA double-labelling experiments. Fifteen days after immunization, most RhoA⁺ cells (brown) co-expressed reactive microglia/macrophages marker ED1 (F, blue), EMAPII (G, blue) or P2X4R (H, blue). The infiltration of W3/13+ T-lympho-cytes (blue) could be detected and cells co-expressed RhoA (brown) (I). Original magnification: A–I x400.