Table 2. Cellular metabolism and culture supernatant cytokine expression levels in EAE splenocytes were significantly higher at disease remission (day 25) than at peak of disease (day 12).
| Isolation day |
12 |
12 |
25 |
25 |
||||
|---|---|---|---|---|---|---|---|---|
| SAgAPLP:LABL ex vivo PLP stimulation |
PBS |
SAgAPLP:LABL |
PBS |
SAgAPLP:LABL |
||||
| Vehicle | 25 µmol/l PLP | Vehicle | 25 µmol/l PLP | Vehicle | 25 µmol/l PLP | Vehicle | 25 µmol/l PLP | |
| Resazurin (ex560/em590) | 1,298 ± 244 | 3,885 ± 745** | 2,012 ± 590 | 6,387 ± 396**,*** | 7,432 ± 244* | 7,212 ± 112 | 6,984 ± 325* | 7,188 ± 161 |
| IL-1α (pg/ml) | 15 ± 5 | 15 ± 5 | 6 ± 4 | 15 ± 8 | 14 ± 5. | 175 ± 26** | 20 ± 8 | 198 ± 25** |
| IL-2 (pg/ml) | 13 ± 6 | 46 ± 22 | 20 ± 8 | 221 ± 55** | 100 ± 23* | 276 ± 112 | 75 ± 4 | 154 ± 65 |
| IL-4 (pg/ml) | ND | 66 ± 22 | 13 ± 4 | 85 ± 15** | 18 ± 3 | 130 ± 10** | 23 ± 8 | 141 ± 9** |
| IL-5 (pg/ml) | ND | ND | ND | 17 ± 11 | 17 ± 11 | 102 ± 9 | ND | 123 ± 10 |
| IL-6 (pg/ml) | 148 ± 131 | 89 ± 26 | 44 ± 30 | 518 ± 155**,*** | 17 ± 7 | 627 ± 112** | 33 ± 5 | 623 ± 147** |
| IL-10 (pg/ml) | ND | 9 ± 4 | ND | ND | ND | 81 ± 12 | ND | 93 ± 12 |
| IL-13 (pg/ml) | ND | 77 ± 67 | ND | 1,505 ± 615*** | 591 ± 461 | 4,305 ± 678** | 494 ± 261 | 4,067 ± 1,041** |
| IL-17 (pg/ml) | ND | 46 ± 12 | ND | 524 ± 236 | 686 ± 657 | 1,833 ± 1,017 | 208 ± 105 | 1,840 ± 580 |
| IL-21 (pg/ml) | ND | ND | ND | ND | ND | 64 ± 15 | ND | 60 ± 17 |
| IL-22 (pg/ml) | ND | 45 ± 32 | ND | 54 ± 10 | 415 ± 323 | 1,713 ± 562** | 538 ± 275 | 1,756 ± 243 |
| IL-27 (pg/ml) | ND | ND | ND | ND | ND | 394 ± 54 | ND | 444 ± 47 |
| IFN-γ (pg/ml) | ND | 83 ± 24 | ND | 699 ± 161**,*** | 120 ± 62 | ≥20,000** | 279 ± 108 | ≥20,000** |
| TNF-α (pg/ml) | 66 ± 24 | 110 ± 29 | 14 ± 5*(P = 0.06) | 160 ± 24** | 191 ± 61 | 247 ± 30 | 274 ± 95* | 288 ± 6 |
| GM-CSF (pg/ml) | 42 ± 26 | 61 ± 39 | 64 ± 26 | 243 ± 56** | 122 ± 11 | 543 ± 74** | 64 ± 37 | 513 ± 110** |
Primary splenocytes were isolated from EAE mice treated with SAgAPLP:LABL or vehicle (PBS) during peak of disease (day 12) or disease remission (day 25). Splenocytes were stimulated with 25 µmol/l PLP or vehicle for 120 hours, and culture supernatant was collected. For cellular metabolism rates, cells were then incubated with resazurin (75 µmol/l final), and fluorescence (ex560/em590) was determined 24 hours later. Data are expressed as mean ± SEM, n = 4–6 mice per group.
Indicates significance (P ≤ 0.05) from untreated day 12 splenocytes treated with PBS in vivo.
Indicates significance (P ≤ 0.05) from respective unstimulated splenocytes.
Indicates significance (P ≤ 0.05) from unstimulated EAE splenocytes isolated from vehicle treated mice during respective disease day.
EAE, experimental autoimmune encephalomyelitis; ex560/em590, 560excitation/590emission; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; ND, not detectable; PBS, phosphate-buffered saline; PLP, proteolipid protein peptide; SAgA, soluble antigen array; SAgAPLP:LABL, SAgA cografted with PLP and LABL peptides; TNF, tumor necrosis factor.