Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12

Abstract

IL-12 is considered a critical proinflammatory cytokine for autoimmune diseases such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-12 is a heterodimer composed of a p35 subunit and a common p40 subunit shared by other cytokines. Both IL-12 p40(-/-) and p35(-/-) mice fail to produce IL-12 p70 heterodimer. However, in contrast to p40(-/-) mice, p35(-/-) mice are highly susceptible to the induction of EAE, establishing that IL-12 p70 is not essential for the development of EAE. When compared with wild-type mice, both p40(-/-) and p35(-/-) mice show deficiencies in primary IFN-gamma responses by lymph node cells. Expression profiling of the inflamed CNS revealed that Th2 cytokines such as IL-4 and IL-10 are upregulated in p35(-/-) mice, whereas LT-alpha and TNF-alpha levels are reduced. These studies show that a molecule other than IL-12 p70, which uses the p40 subunit, fulfills the functions previously attributed to IL-12 with regard to the development and pathogenesis of this autoimmune disease.

Figure 1

IL-12 is not required for clinical disease development. EAE was induced by active immunization of WT (diamonds), p35^-/- (squares) and p40^-/- (circles) mice with MOG_35–55 emulsified in CFA. Clinical disease was monitored and statistical significance assessed using an unpaired Student’s t test. Each data point represents the mean of n animals in one experiment that was repeated at least two more times. ^#P < 0.05, *P < 0.01, comparison to WT mice.

Figure 2

CNS inflammation in IL-12 p35^–/– mice. Animals were sacrificed at day 28 after immunization, and spinal cord inflammation was assessed by hematoxylin and eosin staining. Magnification: ×100. (a) Nonimmunized WT control. (b) Immunized WT mouse. (c) Immunized IL-12 p35^–/– mouse. (d) Immunized IL-12 p40^–/– mouse.

Figure 3

Cellular makeup of infiltrates. Animals were sacrificed at day 24 after immunization, and spinal cord inflammation was assessed by flow cytometry. Infiltrates were stained with antibodies against CD45, CD11b, CD4, CD8, and GR-1 as described in Methods. Gate statistics are expressed as percentages as follows (WT:p35^–/–): R1 (lymphocyte gate CD45^hi/CD11b^–) = 28%:39%; R2 (macrophage/granulocyte gate CD45^hi/CD11b⁺) = 8%:10%; R3 (microglia gate CD45^lo/CD11b⁺) = 30%:25%.

Figure 4

p35^–/– and p40^–/– mice fail to generate Th1 immune responses. (a) Splenocytes from p35^–/–, p40^–/–, and WT mice were unstimulated (black bars) or were stimulated for 48 hours with IFN-γ and LPS (white bars). IL-12 p70 secretion was measured by ELISA. (b–d) p35^–/–, p40^–/–, and WT mice were immunized subcutaneously with KLH in CFA. After 5 days, recall responses of LN cells to either KLH (gray bars) or irrelevant ovalbumin (black bars) were measured by (b) [H]thymidine-uptake, (c) IL-2 ELISA, and (d) IFN-γ ELISA. Shown is a representative of at least two individual experiments.

Figure 5

Expression profile of inflamed spinal cords. RNA was isolated as described from inflamed spinal cords 24 days after immunization. Real-time RT-PCR analysis was performed by measuring amplicon accumulation using SYBR Green dye (white bars, WT; black bars, p35^–/–). Data are expressed as arbitrary units based on the standard curve of triplicate wells ± SD. Values shown are representative of at least three individual experiments. Levels of β-actin mRNA were similar in both samples; other mRNA species levels were normalized to β-actin.