The immunosuppressant FK506 increases GAP-43 mRNA levels in axotomized sensory neurons.

FK506, an immunosuppressant drug used to prevent allograft rejection in organ transplantations, accelerates functional recovery and nerve regeneration in the rat sciatic nerve crush model. While the mechanism by which FK506 increases regeneration is unknown, in contrast to immunosuppression, it does not involve calcineurin inhibition. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique and a digoxigenin-labeled probe, we show that subcutaneous injections of FK506 (10 mg/kg/day) markedly increases the level of axotomy-induced growth-associated protein (GAP-43) mRNA in dorsal root ganglion (DRG) neurons. Quantitation of DRG neurons revealed that FK506 produced a 33% increase in the numbers of neurons exhibiting intense staining. Increased synthesis of GAP-43 may play a role in FK506's ability to speed nerve regeneration.

Oral administration of a nonimmunosuppressant FKBP-12 ligand speeds nerve regeneration.

We recently showed that s.c. injections of a nonimmunosuppressant FK506 binding protein-12 (FKBP-12) ligand (V-10,367) accelerates nerve regeneration in the rat sciatic nerve crush model. Here we examined the oral efficacy of this compound for speeding nerve regeneration. Rats receiving V-10,367 (5, 15 or 50 mg/kg/day) by oral gavage all demonstrated an increase in nerve regeneration compared to vehicle-treated controls. Functional recovery was observed earliest and axonal calibers of regenerating axons in the soleus nerve were largest in the 15 mg/kg group, mean axonal areas being increased by 66% compared to controls. Orally active nonimmunosuppressant FKBP-12 ligands may be useful for the treatment of human peripheral nerve disorders.

A nonimmunosuppressant FKBP-12 ligand increases nerve regeneration.

The immunosuppressant drugs FK506 and cyclosporin A inhibit T-cell proliferation via a common mechanism: calcineurin inhibition following binding to their respective binding proteins, the peptidyl prolyl isomerases FKBP-12 and cyclophilin A. In contrast, FK506, but not cyclosporin A, accelerates nerve regeneration. In the present study, we show that the potent FKBP-12 inhibitor V-10,367, which lacks the structural components of FK506 required for calcineurin inhibition, increases neurite outgrowth in SH-SY5Y neuroblastoma cells and speeds nerve regeneration in the rat sciatic nerve crush model. In SH-SY5Y cells, V-10,367 increased the lengths of neurite processes in a concentration-dependent (between 1 and 10 nM) fashion over time (up to 168 h). Daily subcutaneous injections of V-10,367 accelerated the onset of clinical signs of functional recovery in the hind feet compared to vehicle-treated control animals. Interdigit distances (between the first and fifth digits) measured on foot prints obtained during walking showed an increase in toe spread in V-10,367-treated rats compared to vehicle-treated controls. Electron microscopy demonstrated larger regenerating axons distal to the crush site in the sciatic nerve from V-10,367-treated rats. Quantitation of axonal areas in the soleus nerve revealed a shift to larger axonal calibers in V-10,367-treated rats (400 or 200 mg/kg/day); mean axonal areas were increased by 52 and 59%, respectively, compared to vehicle-treated controls. FKBP-12 ligands lacking calcineurin inhibitory activity represent a new class of potential drugs for the treatment of human peripheral nerve disorders.

Comparative dose-dependence study of FK506 and cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve.

The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al., 1994; Gold et al., 1995). In the present study, we have examined the dose-dependence of FK506's ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1/10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression (i.e., calcineurin inhibition), these results indicate that FK506's nerve regenerative property involves a distinct, calcineurin-independent mechanism.

Stabilization and characterization of antigen-specific T suppressor inducer and T suppressor effector molecules.

H-2 specific T suppressor inducer (Tsfi) and T suppressor effector (Tsfe) factors show a dose-dependent inhibition of one-way mixed lymphocyte responses (MLR) between CBA/J responder spleen cells and C57BL/6 mitomycin C-treated stimulator spleen cells. The hydrophobic proteins Tsfi and Tsfe purified by ammonium sulfate precipitation and affinity methods were stabilized by the addition of Tris-saline pH 8 buffered octylglucopyranoside solution. The stabilized Tsfi and Tsfe fractions stored at 4 degrees C for 3-7 months retained a significant (> 72%) amount of their ability to inhibit MLR. Tsfi and Tsfe purified by salt precipitation and affinity methods were analyzed by SDS-PAGE. Enzyme-linked immunoassay (ELISA) and Western blots indicated that these molecules had T cell receptor (TcR) alpha chain, I-J, and IL-10 epitopes, but not TcR beta chain epitopes.

H-2-specific T suppressor cells. I. Evidence for T suppressor inducer and effector molecules in suppression.

The induction of H-2-specific Ts cells was accomplished by the i.v. injection of X-irradiated (2000R) C57BL/6 spleen cells into CBA/J mice. These Ts cells significantly (78%) suppressed the delayed type hypersensitivity (DTH) response in CBA/J mice injected s.c. with X-irradiated (2000R) C57BL/6 spleen cells and given a footpad challenge with the same cell population 5 days later. We have shown that both CD4 and CD8 T cells were involved in the observed suppression, and these cells secrete T suppressor inducer (Tsfi) and T suppressor effector (Tsfe) molecules. Both Tsfi and Tsfe molecules were shown to significantly inhibit (> 87%) one-way mixed lymphocyte responses between CBA/J and mitomycin C-treated C57BL/6 spleen cells. Using an adoptive transfer method, we showed that mice given both a primary and secondary immunization with X-irradiated C57BL/6 spleen cells to induce H-2-specific Ts cells contain a significantly greater number of Ts cells than mice given only a primary immunization, suggesting the presence of memory Ts cells.

Type D SRV-2 virus-specific CD8+ and CD4- CD8- T cells that regulate virus-induced T cell proliferation in Celebes macaques.

These studies defined SRV-2 envelope peptides 96-102, 127-152, and 233-249 as T cell epitopes that induce significant T cell proliferation. Peripheral blood lymphocytes of Celebes macaques (Macaca nigra) exposed to SRV-2 and currently virus- antibody+, cultured with SRV-2 virus show strongly suppressed T cell responses and have two immunoregulatory T cell populations.