A role for effectors of cellular immunity in epimorphic regeneration of amphibian limbs.

Immune modulation of regeneration of amphibian appendages is suggested, but not proven, by previous studies. Earlier studies have not demonstrated effects of treatments on both epimorphic regeneration and immune responses or restoration of regeneration by specific reversal of immunomodulation. Cyclosporin A (CsA) and interleukin-2 (IL-2) were used in this study to demonstrate the effects of immune suppression and its reversal, on allograft rejection and forelimb regeneration. When administered alone, CsA suppressed rejection of skin allografts and induced a dose-dependent retardation of regeneration. IL-2, administered alone, did not affect allograft rejection or regeneration. However, when combined with CsA, IL-2 abrogated or reversed effects of CsA on both allograft rejection and forelimb regeneration, in a dose-dependent manner. The selective focus of CsA's action and the ability of IL-2 to overcome and reverse these effects strongly suggest that T-lymphocytes participate in or contribute to expression of epimorphic regeneration of amphibian appendages. Further studies are required to better characterize this role.

Differential inflammatory and immunological responses in tissue regeneration and repair.

Repair and regeneration, contrasting resolutions to injury, evoke distinct inflammatory responses. Wound repair evokes a more robust peripheral leukocytosis and is accompanied by a larger inflammatory infiltrate into the wound site. Proliferation of thymocytes and splenocytes was enhanced following injury; however, the ability to be stimulated by myoblasts in coculure is lost by splenocytes harvested from rats undergoing wound repair. These data reflect differences in inflammatory and immunological responses between repair and regeneration and suggest potentially important dialogues occur between injured tissues and inflammatory cells that direct the course of injury resolution.

Cyclic nucleotide metabolism during amphibian forelimb regeneration : II. The protein kinases.

The hypothesis that cAMP mediates neural and endocrine influences on limb regeneration was examined by studying the protein kinases in regenerating limb tissues. Since these enzymes are the vehicles through which cAMP acts intracellularly, an understanding of changes in their concentrations and behaviors during regeneration can be instrumental in elucidating the role of cAMP in this process. Mean activities oscillated throughout regeneration with maximal activities being observed during the mid-late bud stage. The phosphorylation of histone, added to the assay, varied with the stage of regeneration-greatest activity occurring during the early bud stage and very weak activity during the palette and early digital stages. Histone actually appeared to inhibit endogenous phosphorylation during dedifferentiation. In addition, cAMP demonstrated different degrees of enhancement of histone phosphorylation during regeneration-producing its greatest effect at the palette stage and having the least effect at the early bud stage. The results of this study suggest that changes in the absolute amounts of protein kinase are probably not significant in the regulation of regeneration. In addition, the variable acceptability of histone as an exogenous substrate and the variations in the cAMP effects on phosphorylation suggest that physiological changes are occurring in which cAMP might play a significant role. In particular, these data suggest that cAMP might be instrumental in influencing events associated with differentiation and morphogenesis.

Cyclic nucleotide metabolism during amphibian forelimb regeneration : I. The cyclic AMP phosphodiesterases.

Concentrations of the cyclic nucleotides in regenerating limb tissues change in a manner which suggests that they might mediate neural or endocrine influences upon specific developmental events. Since modulation of the role of cAMP within this process can be achieved through cAMP phosphodiesterase, enzymatic activity, relative intracellular distribution, and the kinetic parameters of this enzyme were examined at several stages of limb regeneration in adultNotophthalmus viridescens. Both forms of the phosphodiesterase displayed decreased activity about the time of bud formation. Total phosphodiesterase activity was reduced between 66% and 85% (as compared to intact limbs) between wound healing and palette stages. Relative intracellular distribution (soluble vs. particulate), however, remained essentially constant, 93%-98% soluble for the highK m form and 61%-71% soluble for the lowK m form of the enzyme, throughout this process. The apparentK m of the highK m form increased more than 2-fold during wound healing then fell to approximately 10% (0.7-1.1 µM) of the value of intact limbs (8.3 µM) during dedifferentiation and bud formation. A return to pre-amputational levels was subsequently achieved. In contrast, the apparentK m of the lowK m form increased (from 0.064 to 0.86 µM) during dedifferentiation and began decreasing thereafter. These results are consistent with the hypothesis that one or more mechanisms are operating to modify either the quantity, activity, or physical characteristics of the cAMP phosphodiesterases and that such changes are instrumental in regulating endogenous concentrations of cAMP in limb tissues during regeneration.

Limb regeneration of the adult newt (Notophthalmus viridescens) in the absence of the spleen.

It has been suggested that the immune system might figure prominently in the regulation of forelimb regeneration. However, neither the nature of this influence nor the aspect(s) of regeneration influenced are clearly known. The determination of which components of the immune system are indispensable for regeneration would be a logical first step in attempting to address such questions. This investigation, therefore, examined the effects of removing the spleen, a major lymphoid organ in the newt, upon the progress of regeneration. Splenectomies performed concomitantly with or after forelimb amputation failed to alter the time course of regeneration. Splenectomies, but not sham-splenectomies, performed prior to amputation reduced the time required to achieve successive stages of regeneration under some, but not all conditions, i.e., when performed 10-20 days before amputation, during the late fall and winter. Up until 35 days after amputation, no gross morphological distortions were observed as a result of splenectomy. It was concluded that the spleen is not required for regeneration to occur.

The effects of hypophysectomy upon the endogenous levels of cyclic AMP during forelimb regeneration of adult newts (Notophthalmus viridescens).

Cyclic AMP is believed to play a role in limb regeneration. Using high pressure liquid chromatography, endogenous levels of cyclic AMP in regenerating tissues of normal and of hypophysectomized adult newts were estimated. In normally regenerating limbs, cyclic AMP levels were depressed 7 days after amputation and were elevated at 14 and 21 days. In contrast, limb tissues of hypophysectomized newts displayed elevated cyclic AMP levels at 7, 14 and 21 days after amputation. A correlation exists between depressed levels of cyclic AMP and the occurrence of dedifferentiation and elevated levels of cyclic AMP and morphogenesis. Although elevated cyclic AMP levels later in regeneration might influence morphogenesis, depressed levels of cyclic AMP alone appear inadequate to account for dedifferentiation.