Fig. 4.
Influence of nasal administration of myelin basic protein (MBP) on levels of IFN-γ-secreting Th1-like splenocytes. Splenocytes were prepared from killed rats and ELISPOT assays were carried out as described in Materials and Methods. Using our protocol, control wells without added MBP or wells with the irrelevant control antigen acetylcholine receptor (AChR) showed very few spots (<5 per 105 splenocytes), and were subtracted from numbers of IFN-γ-secreting MBP-reactive cells. Results are expressed as mean numbers from five rats ± s.d. (a) Rats were pretreated nasally either with PBS (□) or 600 μg/rat of MBP (▪) before induction of EAE as scheduled in Materials and Methods. Groups of five rats were killed on day 13 or day 60 p.i. Strongly reduced numbers of MBP-reactive IFN-γ-secreting splenocytes were recorded at both time points in rats pretreated with MBP versus those treated with PBS nasally. (b) EAE was induced in DA rats by immunization with spinal cord homogenate (SCH) + Freund's incomplete adjuvant (FIA), followed by nasal administration of either PBS or 60 μg/rat of MBP from day 9 p.i. and for 10 consecutive days. Groups of five rats were killed at different time points during or after this treatment, and numbers of MBP-reactive IFN-γ-secreting splenocytes were evaluated by ELISPOT. No difference of numbers of MBP-reactive IFN-γ-secreting cells was observed between MBP-treated (▪) and PBS-treated control rats (□) on day 26 p.i., i.e. 1 week after treatment was stopped. However, higher numbers of MBP-reactive IFN-γ-secreting splenocytes were observed in MBP-treated rats (▪) when examined on days 11 and 13 p.i., i.e. during the early phase of MBP treatment, versus control rats (□). (c) EAE was induced by immunization of DA rats with SCH + FIA, followed by administration of either PBS or 160 μg/rat of MBP nasally from day 9 p.i. and for 10 consecutive days. Groups of five rats were killed at different time points during (day 11 p.i.) or after (day 26 p.i.) treatment, and numbers of MBP-reactive IFN-γ-secreting splenocytes were evaluated. Higher numbers of IFN-γ-secreting cells on day 11 p.i. (early phase of MBP treatment) but lower numbers of such cells on day 26 p.i. (1 week after MBP treatment was stopped) were observed in MBP-treated versus PBS-treated control rats. ***P< 0.001; **P< 0.01; *P< 0.05.