Enzo Ottaviani

Inflamm‐aging: An Evolutionary Perspective on Immunosenescence

Abstract: In this paper we extend the “network theory of aging,” and we argue that a global reduction in the capacity to cope with a variety of stressors and a concomitant progressive increase in proinflammatory status are major characteristics of the aging process. This phenomenon, which we will refer to as “inflamm‐aging,” is provoked by a continuous antigenic load and stress. On the basis of evolutionary studies, we also argue that the immune and the stress responses are equivalent and that antigens are nothing other than particular types of stressors. We also propose to return macrophage to its rightful place as central actor not only in the inflammatory response and immunity, but also in the stress response. The rate of reaching the threshold of proinflammatory status over which diseases/disabilities ensue and the individual capacity to cope with and adapt to stressors are assumed to be complex traits with a genetic component. Finally, we argue that the persistence of inflammatory stimuli over time represents the biologic background (first hit) favoring the susceptibility to age‐related diseases/disabilities. A second hit (absence of robust gene variants and/or presence of frail gene variants) is likely necessary to develop overt organ‐specific age‐related diseases having an inflammatory pathogenesis, such as atherosclerosis, Alzheimers disease, osteoporosis, and diabetes. Following this perspective, several paradoxes of healthy centenarians (increase of plasma levels of inflammatory cytokines, acute phase proteins, and coagulation factors) are illustrated and explained. In conclusion, the beneficial effects of inflammation devoted to the neutralization of dangerous/harmful agents early in life and in adulthood become detrimental late in life in a period largely not foreseen by evolution, according to the antagonistic pleiotropy theory of aging.

Cytotoxicity and cytotoxic molecules in invertebrates

Although lacking the components that characterize the acquired immunity systems of vertebrates, invertebrates nevertheless possess effective general innate immune mechanisms which exhibit striking parallels with those of vertebrates. These innate immune systems include both cellular and humoral elements. Invertebrate phagocytes synthesize both oxygen‐dependent and oxygen‐independent molecules to combat infectious agents. Cytotoxic substances employed by invertebrates include reactive intermediates of oxygen and nitrogen, antimicrobial peptides, lectins, cytokine‐ and complement‐like molecules, and quinoid intermediates of melanin. The signal transduction pathways that are involved in mediating the production of these substances appear to be very similar among animal species, suggesting a common ancestral origin for the innate immune systems. BioEssays 22:469—480, 2000.

The invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems

Abstract In invertebrates, lower vertebrates and higher vertebrates, a common pool higher conserved molecules appears to mediate phagocytosis the stress response and inflammation. Here Enzo Ottaviani and Clendio Franceschi propose an evolutionary hypothesis based on an integrated network of adaptive mechanisms that are critical for survival. The macrophage plays a pivotal role in this process.

Evidence for nitric oxide production and utilization as a bacteriocidal agent by invertebrate immunocytes

The present study demonstrates that molluscan immunocytes are able to produce a chemical bacteriocidal substance which can be indirectly identified as nitric oxide (NO). The cells were analyzed in vitro on slides using computer-assisted microscopic image analysis to detect changes in cell conformation as well as to quantify the number of bacteria present. Sodium nitroprusside yields NO in solution causing bacterial clumping. The same phenomenon occurs in the presence of invertebrate immunocytes. Escherichia coli lipopolysaccharide also increases the number of bacteria found around the immunocytes, but this effect is selectively prevented by the addition of inhibitors of nitric oxide synthase, suggesting that this bacterial clumping is caused by the cells liberating NO. Interestingly the cells presumably producing NO maintain a round morphology. These findings suggest that immunocytes are able to kill bacteria by two mechanisms, i.e., phagocytosis and NO production.

Pro-Opiomelanocortin-Derived Peptides, Cytokines, and Nitric Oxide in Immune Responses and Stress: An Evolutionary Approach

In vertebrates, including man, the study of stress has contributed substantially to unravelling the complex relationship between immune-neuroendocrine interactions and the systems involved. On the basis of data on the presence and distribution of the main actors (POMC products, cytokines, biogenic amines, and steroid hormones) in different species and taxa from invertebrates to vertebrates, we argue that these responses have been deeply connected and interrelated since the beginning of life. Moreover, the study of nitric oxide suggests that the inflammatory reaction is located precisely between the immune and stress responses, sharing the same fundamental evolutionary roots. The major argument in favor of this hypothesis is that the immune, stress, and inflammation responses appear to be mediated by a common pool of molecules that have been conserved throughout evolution and that from a network of adaptive mechanisms. One cell type, the macrophage, appears to emerge as that most capable of supporting this network critical for survival; it was probably a major target of selective pressure. All these data fit the unitarian hypothesis we propose, by which evolution favors what has been conserved, rather than what has changed, as far as both molecules and functions are concerned.

Nitric oxide synthase activity in molluscan hemocytes

The hemocytes of the freshwater snail Viviparus ater have nitric oxide synthase (NOS) activity, as demonstrated by [3H]citrulline and nitrite+nitrate formation. The enzyme is NADPH dependent and is competitively inhibited by the mammalian NOS inhibitor (K i = 4.7 μM). The K m for l‐arginine is 2.5 μM. 70% of the total activity is observed at very low free Ca2+ concentration (3 nM). LPS treatment increased total NOS activity 2.4 fold. The activity is partly present in the non‐soluble fraction of hemocytes (24% and 8% in non‐stimulated and LPS‐stimulated snails, respectively). An antiserum to the C‐terminal synthetic pentadecapeptide of the rat cerebellar NOS inhibited the enzyme activity in a concentration‐dependent manner. This is the first biochemical demonstration of the existance of NOS activity in molluscan hemocytes, the cells responsible for defence mechanisms.

Presence of ACTH and β-endorphin immunoreactive molecules in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) and their possible role in phagocytosis

The presence of ACTH and beta-endorphin immunoreactive molecules in the cell-free hemolymph and in the hemocytes of the freshwater snail Planorbarius corneus were demonstrated by immunocytochemistry and RIA tests. Only spreading phagocytic hemocytes were positive, in contrast with other hemocytes devoid of phagocytic activity, i.e., round hemocytes. These data were confirmed by flow cytometry. Another cell type with marked phagocytic activity, i.e., digestive cells of digestive gland, were also positive to anti-ACTH. Corticotropin-releasing factor immunoreactive molecules were found in the cell-free hemolymph and hemocytes, by RIA. Our data suggest that cells with phagocytic activity, the oldest immune response, may represent a suitable model to unravel the tangled web of the common ancestor of the immune and the neuroendocrine systems.

Cytokines and invertebrate immune responses.

A profound interrelationship between cytokines and invertebrate (molluscs) immune responses has been reported. Different cytokines (IL-1 alpha, IL-2 and TNF-alpha) significantly stimulate molluscan hemocyte motility, increasing phagocytic activity and provoking the induction of nitric oxide synthase. As far as cell motility is concerned, the response to different cytokines varied between species. These and other recently reported findings (Ottaviani et al (1994) FEBS Lett 351, 19-21; Ottaviani et al (1995) Biochem Biophys Res Commun 207, 288-292) suggest that cytokines are important, ancestral, and functionally conserved molecules, which have also maintained their pleiotropicity, redundancy in the mode of action, and high promiscuity of their receptors during evolution.

Stress response in the freshwater Snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) : interaction between CRF, ACTH, and biogenic amines

Previous studies reported that ACTH molecules influence chemotactic and phagocytic activities of hemocytes in the freshwater snail, Planorbarius corneus. The present study reveals that ACTH and CRF affect the release of biogenic amines. Hemocytes from P. corneus hemolymph incubated in vitro with ACTH for 15, 30, and 45 min released epinephrine, norepinephrine, and dopamine. The greatest release occurred after 15 min, while after 45 min the values were similar to those of the controls. Similar incubations with CRF also provoked a release of biogenic amines, this being mainly mediated by the release of endogenous ACTH. These data suggest that (i) ACTH and CRF provoke the release of biogenic amines; (ii) there is a direct relationship between CRF, ACTH, and biogenic amines, with the hemocytes as the target; (iii) exogenous ACTH can mimic an ancestral type of stress response; (iv) the major pathway of the stress response in P. corneus is mediated by a CRF-ACTH-biogenic amine axis. These data should help to unravel part of the complex molecular signaling mechanisms involved in the physiological/endocrinological reaction of invertebrate organisms to stress, and suggest that a stress response unexpectedly similar to that present in mammalian cells is detectable in invertebrates.

The blood cells of the freshwater snail Planorbis corneus (Gastropoda, pulmonata)

A study of the morphology and behaviour (cell adherence and spreading, in vitro phagocytosis and rosette formation) of blood cells of Planorbis corneus, revealed the presence of two cell types: the spreading haemocyte and the round haemocyte. Results obtained with vinblastine confirmed that these cell forms are distinct cell types and not different maturational stages of a single cell type.