Mario Marini

Positive and negative immunomodulation by opioid peptides

The data that follow review part of the existing evidence concerning the neuroimmune functions mediated by opioid peptides, with particular regard to dual immunomodulatory effects. Limited references to substances other than opioid peptides are included, mainly to emphasize the possible similarities in the mediation of neuroimmune interactions by different informational substances, while the interactions directed from the immune to the nervous system have deliberately been omitted.

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.

Hydrolysis and binding of leucine enkephalin to lymphomic and erythroleukaemic cell lines

Hydrolysis and binding of labelled leucine enkephalin have been measured in the presence of cell lines of lymphoid and erythroid origin. The radioactive label was found to be associated to all lines studied. In the presence of these cells, enkephalin is rapidly hydrolyzed, forming three tyrosine-containing peptides: Tyr, Tyr-Gly and Tyr-Gly-Gly. Conversely, the presence of selective protease inhibitors reduces both enkephalin degradation and binding. Data obtained suggest the involvement in enkephalin hydrolysis of aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In addition, they suggest that the radioactive label associated to cells can be related to the peptides formed by the enzyme degradation of enkephalin and not to the intact pentapeptide.

Interindividual variability of enkephalin-degrading enzymes in human plasma.

The interindividual variability of the hydrolysis of leucine enkephalin, and of the formation of its hydrolysis by-products has been studied in human plasma. In agreement with known data, the data obtained indicate that Leu-enkephalin is degraded by several enzymes, belonging to three classes: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The relative ratio of the substrate degraded by each enzyme class-as well as the expression of the single enzyme species within each class-appears to be individually determined. Interindividual variability observed seems controlled by two main factors: the pattern of enkephalin-degrading enzymes and, more notably, the low molecular weight plasma inhibitors. Both these factors appear to be partially specific of each donor. Possibly because of the composition of these factors, the hydrolysis pattern of the substrate is characteristic of each donor, and constant in blood obtained from successive drawings, at least within a relatively short period of time.

Enzymes and inhibitors in leu-enkephalin in metabolism in human plasma

The enzymes degrading leucine enkephalin in human plasma and the inhibitors active on these enzymes were studied by kinetic and chromatographic techniques. Data obtained evidence the existence of complex kinetics of leu-enkephalin hydrolysis and of formation of its hydrolysis byproducts. These appear to originate from the combined effect of further hydrolysis of the enkephalins fragments after their release and of competition between the different enzymes present in plasma. Chromatographic separation of plasma proteolysis inhibitors indicates the existence of several pools of substances acting on all three enzyme groups that degrade leu-enkephalin. The partial specificity of these substances induces competition effects: consequently, the actual protection over leu-enkephalin is considerably lower that the total inhibitory activity. That notwithstanding, plasma inhibitors control enkephalin hydrolysis to a relevant extent, while they modify only slightly the ratio of hydrolysis between the different enzymes. This latter parameter—and specifically the large prevalence of aminopeptidases over dipeptidylaminopeptidases and dipeptidylcarboxypeptidases—appears controlled mainly by kinetic factors.

Soluble enkephalin-degrading enzymes released by lymphomic and erythroleukaemic cell lines

The possible existence of soluble proteolytic enzymes released by cells of lymphomic (U937 and 1301) and erythroleukaemic (K562) lines was studied measuring the hydrolysis of3H-leucine enkephalin in the presence of cell-free supernatants obtained from these lines. Results indicate that leu-enkephalin is rapidly degraded in the presence of these supernatants, and that enkephalin disappearance is paralleled by the formation of peptides that can be interpreted as its hydrolysis fragments. To characterize the factors involved in leu-enkephalin degradation, cell supernatants were analyzed by ion exchange and by steric exclusion chromatography. Data obtained indicate the presence of three groups of proteins active in leu-enkephalin degradation: aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In all three lines, these enzymes are represented by a considerable number of distinct activities. The sizable number of soluble enzymes identified and the signficant total activity observed suggest a possible role in the regulatory degradation of informational peptides, as proposed by several groups for the membrane-bound proteolytic enzymes of immunocompetent cells.

Hydrolysis and association of leucine enkephalin to lymphomic and erythroleukaemic cell lines. III: Soluble enzymes

The possible existence of soluble proteolytic enzymes released by cells of lymphomic (U937 and 1301) and erythroleukaemic (K562) lines was studied measuring the hydrolysis of 3H-leucine enkephalin in the presence of cell-free supernatants. Results obtained indicate that in the presence of these supernatants leu-enkephalin rapidly disappears and that enkephalin disappearance is paralleled by the formation of peptides that can be interpreted as its hydrolysis fragments. Chromatographic analyses of cell supernatants indicate the presence of three groups of proteins active in leu-enkephalin degradation: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In all three lines, soluble enzymes are represented by a sizeable number (from 13 to 25) of distinct activities. The number of enzymes identified and their considerable total activity suggest a possible role in the regulatory degradation of informational peptides, as proposed by several groups for the membrane-bound proteolytic enzymes of immunocompetent cells.

Hydrolysis and association of leucine enkephalin to lymphomic and erythroleukaemic cell lines--II.

The relationship between the hydrolysis of labelled leucine enkephalin and the association of its radioactive label to the cells of lymphomic and erythroleukaemic cell lines have been studied using intact cells and resealed membranes obtained from these cells as models. Hydrolysis by cell enzymes and its effect on association have been analysed using protease inhibitors and non-hydrolysable enkephalin analogues. Results obtained confirm that hydrolysis of the pentapeptide is a prerequisite for association of the radiolabel to cells. The same results provide evidence of marked differences between enkephalin hydrolysis by whole cells and hydrolysis measured in the presence of resealed membranes, suggesting the existence in intact cells of proteolytic enzymes other than those bound to the membranes. The lack of reversibility of association and the intracellular localization of the radioactive label suggest that the association measured is prevailingly caused by internalization of a hydrolysis fragment, and not by binding to receptors. In order to determine the nature of the active fragment, association was measured in the presence of all four labelled N-terminal hydrolysis fragments of leu-enkephalin under conditions of nearly-total inhibition of proteolytic enzymes. Under these conditions, the label carried by Tyr, but not that carried by the other N-terminal fragments, was associated with cells. Free Tyr, furthermore, inhibits the association to cells of both labelled Tyr and leu-enkephalin. Data summarized above are consistent with the hypothesis that the radioactive label is taken up by the cells as Tyr, freed from the parent peptide by cell-related enzymes. The same data tend to exclude that a relevant fraction of the intact pentapeptide is bound to membrane receptors or that the radioactive label is carried into the cell by a N-terminal fragment other than Tyr.

Role of hydrolysis in the association of leu-enkephalin to peripheral blood mononucleate cells.

The possible hydrolysis of labelled leucine enkephalin in the presence of peripheral blood mononucleate cells and its association with the same cells have been investigated. Results obtained indicate that under experimental conditions leu-enkephalin is rapidly hydrolysed in the presence of model cells. Under the same conditions, the radioactive label is partially associated with the cells. The presence of protease inhibitors reduces both enkephalin degradation and association of the radioactive label with the cells. The hypothesis that the associated species is intact leu-enkephalin is not supported by the relative kinetics of enkephalin hydrolysis and of association of the radioactive label with the cells, nor by the statistically significant correlation between these data, nor again by the effect of proteolysis inhibitors. The same data agree with the hypothesis that the radioactive label associated with the cells is represented by one, or several, of the fragments formed by enkephalin hydrolysis, not by the intact molecule. However, results obtained in the presence of hydrolysis-stable DADL indicate association with the cells of the intact peptide. Moreover, antigen stimulation has opposite effects on the two peptides: with leu-enkephalin, hydrolysis and association are increased, with DADL association is decreased by stimulation. The sum of these data seems to suggest the co-existence in PBMC of two phenomena, i.e. internalization of one, or several, of the fragments formed by the enzyme degradation of enkephalin and binding to membrane receptors of the intact pentapeptide.

Effect of stimulation on soluble proteolytic enzymes released by peripheral blood mononuclear cells

The release of soluble peptidases active in the degradation of leucine enkephalin by human peripheral blood mononuclear cells (PBMC), and the effect of stimulation of mononuclear cells on the release of these enzymes are reported. Results obtained indicate that leu-enkephalin is partially degraded in the presence of soluble supernatants prepared from mononuclear cells. In accord with data previously obtained with immunocompetent cell lines, three classes of enzymes appear to be involved in leu-enkephalin hydrolysis: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. Phytohemagglutinin stimulation of mononuclear cells appears to cause a relevant increase of the total activity of the soluble enzymes, as well as a partial rearrangement of the ratio between the different enzyme activities. A similar effect is also evident upon chromatographic separation of the soluble supernatants: the relative activity of the several distinct enzymes--notably aminopeptidases and dipeptidylaminopeptidases--identified after column separation is significantly modified by PBMC stimulation. The effect of stimulation of mononuclear cells on the release of soluble enzymes can be interpreted as an indication of the role of these enzymes--possibly in the regulative process of informational peptides--similar to that described in the case of membrane enzymes.