The cytokine interleukin-6 is sufficient but not necessary to mimic the peripheral conditioning lesion effect on axonal growth

Abstract

Lesioning the peripheral branch of a dorsal root ganglion (DRG) neuron before injury of the central branch of the same neuron enables spontaneous regeneration of these spinal axons. This effect is cAMP and transcription dependent. Here, we show that the cytokine interleukin-6 (IL-6) is upregulated in DRG neurons after either a conditioning lesion or treatment with dibutyryl-cAMP. In culture, IL-6 allows neurons to grow in the presence of inhibitors of regeneration present in myelin. Importantly, intrathecal delivery of IL-6 to DRG neurons blocks inhibition by myelin both in vitro and in vivo, effectively mimicking the conditioning lesion. Blocking IL-6 signaling has no effect on the ability of cAMP to overcome myelin inhibitors. Consistent with this, IL-6-deficient mice respond to a conditioning lesion as effectively as wild-type mice. We conclude that IL-6 can mimic both the cAMP effect and the conditioning lesion effect but is not an essential component of either response.

Figure 1.

IL-6 increases in response to either elevated cAMP or a conditioning lesion. A, Dissociated DRG cells were either treated in culture for 18 h with 1 mm db-cAMP or removed from the animal 18 h after a peripheral lesion (CondLesion), and mRNA was isolated and subjected to either microarray analysis or Q-RT-PCR for IL-6. The control was mRNA from untreated, unlesioned animals. Each experiment was performed at least three times. B, Radiolabeled consensus CRE (lanes 1–4) and IL-6 CRE (lanes 5–8) probes were incubated with 25 ng (lanes 2 and 6), 50 ng (lanes 3 and 7), and 100 ng (lanes 4 and 8) of PKA-phosphorylated recombinant CREB. C, Unlabeled, annealed oligomers representing the CRE consensus (CRE wt) and IL-6 CRE (IL-6 wt) sequences were added in 50-fold (lanes 3 and 8), 100-fold (lanes 4 and 9), and 200-fold (lanes 5 and 10) excess over labeled CRE consensus or IL-6 CRE probes to compete for binding of 50 ng of PKA-phosphorylated recombinant CREB (lanes 2–5 and 7–12). A mutated IL-6 CRE sequence (IL-6 mt) was also used to compete for binding of protein at 50-fold (lane 11) and 100-fold (lane 12) excess over labeled probe. D, DRG cells were cultured with 1 mm db-cAMP for the times indicated, after which the cells were lysed and subjected to immunoblotting for IL-6. Recombinant IL-6 (rIL-6) was used as a positive control. E, Hippocampal neurons (HN) or DRG neurons were cultured with (▧) or without (▪) 1 mm db-cAMP for 24 h, after which time the supernatants were collected and subjected to an ELISA for IL-6. Results are expressed as the picomole of IL-6 secreted from 10⁶ cells in that time. Error bars indicate SE of at least three experiments.

Figure 2.

Exogenous IL-6 overcomes inhibition by MAG and myelin in a dose-dependent manner. Isolated P5 rat pup DRG neurons (A, C) or hippocampal neurons (HN; B, D) were dissociated and cultured on MAG-expressing CHO cells (▧; A, B), on control CHO cells (▪; A, B), or on a substrate of myelin (▧; C, D) or poly-l-lysine (▪; C, D) in the presence or absence of various concentrations of IL-6 overnight before being fixed and stained for β-III tubulin. Results represent the average length of the longest neurite from 180 to 200 neurons ± SEM from at least three experiments.

Figure 3.

IL-6 overcomes inhibition in a transcription-dependent, cAMP-independent manner via the IL-6R/gp130 receptor complex. DRG neurons (A, C) or hippocampal neurons (HN; B, D) were plated onto either MAG-expressing (▧) or control CHO cells (▪) in the presence or absence of IL-6 (20 ng/ml), a PKA inhibitor (KT5720, 200 nm), an inhibitor of transcription (DRB, 5 μm), a JAK inhibitor (AG490, 0.2 or 2 μm), a soluble form of gp130 (gp130-Fc; 2, 10, or 50 μg/ml), or an antibody to gp130 (anti-gp130, 10 or 50 μg/ml) as indicated and cultured overnight before being fixed and immunostained for β-III tubulin. Results represent the average length of the longest neurite from 180 to 200 neurons ± SEM from at least three experiments.

Figure 4.

IL-6 is not necessary for db-cAMP or a conditioning lesion to overcome inhibition. DRG neurons from unlesioned animals (A) or from animals 1 week after a conditioning peripheral lesion (B) were plated onto either MAG-expressing (▧) or control (▪) cells and cultured overnight with or without db-cAMP (1 mm) in the presence or absence of AG490 (0.2 or 2 μm), gp130-Fc (2, 10, or 50 μg/ml), or an antibody to gp130 (10 or 50 μg/ml) as indicated. The cultures were fixed and stained for β-III tubulin. Results represent the average length of the longest neurite from 180 to 200 neurons ± SEM from at least three experiments.

Figure 5.

IL-6 is not necessary for the conditioning lesion effect on dorsal column regeneration. Representative sagittal sections of the dorsal spinal cord showing CTb-immunolabeled axons through the lesion site are shown. Many regenerating axons attempting to grow through and beyond the lesion site were observed in both wild-type and IL-6−/− mice (A, B, arrows). Without a previous conditioning lesion, most injured axons retracted from the injury site and formed dystrophic ends in both wild-type (C) and IL-6−/− (D) mice. Asterisks indicate the lesion site in the dorsal cord.

Figure 6.

Quantitation of axonal regeneration in IL-6-deficient and wild-type mice. The extent of axonal regeneration was assessed by quantifying the CTb-immunolabeled fibers along the path of axon growth in the dorsal column using MetaMorph image analysis software. There was no significant difference in the extent of regeneration of the injured dorsal column axons between wild-type and IL-6−/− mice after a peripheral conditioning (Cond) lesion.

Figure 7.

Intrathecal delivery of IL-6 overcomes inhibition by MAG when DRG neurons are subsequently cultured. IL-6, at the doses indicated, was delivered to P60 rats for 24 h before the DRG neurons were removed and cultured on either MAG-expressing (▧) or control (▪) CHO cells for 24 h before being fixed and stained for β-III tubulin. Results represent the average length of the longest neurite from 180 to 200 neurons ± SEM from at least three animals under each condition.

Figure 8.

Intrathecal delivery of IL-6 is sufficient to promote dorsal column axon regeneration in vivo. A DCL was performed on P60 rats at T6/7 and, at the same time, intrathecal mini-pumps were inserted delivering IL-6 at a dose of 2 pmol/kg/h (A) or saline (B) for 2 weeks. After an additional 4 weeks, the animals were perfused and sectioned. Five days before the animals were killed, dorsal column axons were retrogradely traced with HRP. The images are longitudinal sections of thoracic spinal cord. The asterisk indicates the lesion center.