Massimo De Martinis

Inflamm-ageing and lifelong antigenic load as major determinants of ageing rate and longevity

Immunosenescence is the consequence of the continuous attrition caused by chronic antigenic stress. The most important characteristics of immunosenescence (accumulation of memory and effector T cells, reduction of naive T cells, shrinkage of T cell repertoire, reduction of the immunological space) are compatible with this assumption. Immunosenescence can be taken as proof that the beneficial effects of the immune system, devoted to the neutralization of harmful agents early in life, become detrimental late in life, in a period not foreseen by evolution. This perspective could explain the mechanisms of the ageing process as well as the pathogenesis of age‐related diseases.

Immunosenescence and infectious diseases.

Infectious diseases are major causes, with malignancies, of morbidity and mortality in the elderly. Increased susceptibility to infections may result from underlying dysfunction of an aged immune system; moreover, inappropriate immunologic functions associated with aging can determine an insufficient response to vaccines. Impairments of cellular, humoral and innate immunity in the elderly, contributing to increased incidence of infectious diseases, are discussed in this review.

The Immune System in the Elderly

Apoptosis is a complex cell-death process that allows cells to die in a controlled fashion. Our overall health relies to a great extent on the proper balance between the normal removal of damaged or unneeded cells via apoptosis and proliferation of the cells that comprise our body. Increasing evidence suggests that apoptosis is involved in many physiological processes and pathological conditions. It plays an important role during development, in maintaining tissue homeostasis, in responding to cellular damage, and in preventing neoplastic diseases. Apoptosis is a key regulator of clonotypic diversity generation during lymphocyte ontogenesis and is essential for the proper function of the immune system, controlling lymphocyte activation and clonal expansion following antigenic stimulation. There are various types of apoptosis, induced by different kinds of stimuli and in cells and tissues of different natures. On the basis of the nature of the apoptosis-inducing stimuli, two main apoptotic pathways can be identified: an activation-induced apoptosis, initiated by a variety of signals, such as the binding of ligands to their death-promoting receptors on the cell surface, and a damage-induced apoptosis, triggered by a damage to the nucleous or other cellular components. Apoptosis is markedly involved in many changes characteristic of immunosenescence, such as thymic involution, alteration of T-cell repertoire, accumulation of memory/effector cells, and autoimmunity. The intense investigation of the age-related changes occurring in cell-death phenomena and on their precise impact on aging has resulted in controversial data. During senescence, the activation-induced and damage-induced apoptotic pathways could be differentially modulated, with variable impacts on the aging process. Changes in either of these two main apoptotic networks that may occur during aging could lead to disease. A correct modulation of apoptosis may be useful for prolonging the lifespan or at least reducing age-related degenerative and inflammatory pathologies and neoplastic diseases whose incidence increases with age. Careful study of literature together with personal experience in the field of senescence causes us to propose a new reading register that better explains and integrates many of the apparently discordant results.

Osteoporosis, Inflammation and Ageing

Osteoporosis is a condition characterized by low bone mass and increased bone fragility, putting patients at risk of fractures, which are major causes of morbidity substantially in older people. Osteoporosis is currently attributed to various endocrine, metabolic and mechanical factors. However, emerging clinical and molecular evidence suggests that inflammation also exerts significant influence on bone turnover, inducing osteoporosis. Numerous proinflammatory cytokines have been implicated in the regulation of osteoblasts and osteoclasts, and a shift towards an activated immune profile has been hypothesized as important risk factor. Chronic inflammation and the immune system remodelling characteristic of ageing, as well as of other pathological conditions commonly associated with osteoporosis, may be determinant pathogenetic factors. The present article will review the current perspectives on the interaction between bone and immune system in the elderly, providing an interpretation of osteoporosis in the light of inflamm-ageing.

Phenotypic and functional changes of circulating monocytes and polymorphonuclear leucocytes from elderly persons

The function and phenotype of monocytes and granulocytes in the elderly is consistently remodelled. Because leucocyte adhesion molecules play important roles in mediating a wide variety of leucocyte functions, age‐related changes in their expression on granulocyte and monocyte surfaces could be partially responsible for immune dysfunctions during senescence. Considering the central role of innate immunity in the process of immunosenescence and the involvement of cell adhesion molecules (CAM) in the great majority of leucocyte functions, we studied the expression of CD50 and CD62L adhesion molecules in peripheral blood granulocytes and monocytes from healthy elderly and young subjects. We show here that the percentage of granulocytes and monocytes expressing CD62L is decreased in the elderly, whereas its density expression is unchanged on both cell types. A downregulation of the density expression of CD50 at a per cell level characterizes granulocytes in the elderly, whereas CD50 expression on monocytes from old subjects shows a peculiar attitude: its density expression decreases whereas the number of positive cells is expanded. The downregulation of this receptor on granulocytes from aged people could determine a state of hyperactivation contributing to the proinflammatory status of the elderly, while the lower expression on monocytes could therefore contribute to the impaired antigen presentation in the elderly. On the other hand, the increased number of CD50 positive monocytes in the elderly, despite its decreased density expression at a per cell level, could be interpreted as an attempt to counteract the inability to mount strong immune responses. Both CD50 and CD62L changes in ageing polymorphonuclear (PMN) cells allow recognition as non‐self or senescent self to permit macrophages in the liver and spleen to remove them from the circulation. The increased proportion of granulocytes and monocytes lacking CD62L and the downregulation of CD50 intensity expression on both cell types may suggest a state of in vivo activation. Therefore, CD50 and CD62L shedding from the cell surface of activated granulocytes and monocytes could be interpreted as a tentative to counteract the dangerous effects of an excessive chronic inflammation in the elderly. However, the increased proportion of CD62L negative granulocytes in the elderly leads to an impairment in cell adhesion which is the first line of response to acute inflammatory stimuli. This phenomenon likely contributes to the increased susceptibility to acute infections of elderly people.

Immunophenotypical Changes of T Lymphocytes in the Elderly

Background: Substantial changes in both representation and function of T lymphocyte subsets have been reported with advancing age. However, till now, no systematic studies focused on age-dependent changes in the expression intensity of the major T lymphocyte surface receptors. Objective: The present study was undertaken in order to establish age-related differences in lymphocyte subpopulations by simultaneously measuring three surface antigens in young and elderly people. Method: Peripheral blood T cell subsets from 20 healthy elderly individuals and 15 healthy young adult donors were examined by means of a quantitative three-color flow cytometry method. Results: Activated (HLA-DR+) and memory (CD45RO+) T cells, CD3+CD7– T lymphocytes, and cells expressing natural killer (NK) markers (CD3–CD56+ NK cells and CD3+CD56+ T lymphocytes) were expanded, whereas T lymphocytes expressing the adhesion molecule CD62L were lower in elderly compared with young donors. In addition to alterations in the percentages of T cell subsets during senescence, several changes in the intensity expression of T cell antigens were also detected. CD3 antigen expression was downregulated on total T lymphocytes as well as on the memory T cell subset, while CD56+ T cells exhibited increased CD3 levels. Moreover, CD2 expression, unchanged on NK cells, was upregulated on T lymphocytes from elderly subjects. CD3+CD7– T cells exhibited increased expression of CD8 antigen, while the intensity expression of HLA-DR on activated T cells and CD7 on both T and NK lymphocytes was decreased. T cells from elderly subjects also exhibited higher expression of CD50 and CD62L adhesion molecules as compared with young ones. Conclusion: These T cell antigen expression modulations during senescence, in addition to the alteration in the frequency of the various T lymphocyte subsets, could contribute to the complex remodeling of the immune function characteristic of the elderly.

Coagulation and cancer: implications for diagnosis and management.

Coagulation disorders are a common problem in neoplastic patients and many factors contribute to increase the risk of thromboembolic events in these patients. An hypercoagulable state is induced by malignant cells interacting directly with hemostatic system and activating the coagulation cascade. More sensitive tests to assess an hypercoagulable state in cancer patients have been developed; even though these tests are always altered in cancer patients, none of them possess a clinical significance in terms of predictive value for the occurence of thromboembolism and disease prognosis in the individual patient. The most frequent thromboembolic complications in cancer patients are deep vein thrombosis of the lower extremities and pulmonary embolism; therefore, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura or haemolytic uremic syndrome are special manifestations of neoplastic disease. Diagnosis of idiopathic deep vein thrombosis, in the absence of other risk factors, could indicate the presence of occult malignant disease; however, the need for an extensive work-up to detect malignancy is still controversial. Neoplastic patients showing a thromboembolic event should be treated with unfractioned heparin or, alternatively, with low molecular weight heparins. In order to prevent recurrence, the administration of heparin should be associated and followed by an oral anticoagulant drug. In recent years new approaches in anti-aggregation therapy have been studied, such as COX-inhibitors, cicaprost and ReoPro; further studies are needed to determine the usefulness of these molecules in treatment of malignancies.

Apoptosis Remodeling in Immunosenescence: Implications for Strategies to Delay Ageing

Immunosenescence is characterized by a peculiar remodeling of the immune system, mainly induced by lifelong antigenic burden and oxidative stress. Apoptosis or programmed cell death plays a central role in the ageing process. Both recurrent antigenic stimulations and oxidative metabolism by-products, impinging upon the immune system, modify the apoptotic capability of lymphocytes, driving immunosenescence. The apoptosis remodeling, in addition to inflamm-ageing, i.e. the upregulation of anti-stress responses and inflammatory cytokines, represents one of the major determinants of ageing rate and longevity, as well as of the most common age-related diseases. The cells of the immune system undergo two different kinds of apoptotic processes: activation-induced cell death (AICD), geared towards the elimination of unnecessary lymphocytes following clonal expansion, and damage-induced cell death (DICD), particularly important for preventing the onset of neoplastic proliferations. During senescence these apoptotic pathways are differentially modulated, with variable impacts on the ageing process. A correct modulation of apoptosis may be useful for prolonging the lifespan or at least reducing age-related degenerative, inflammatory and neoplastic diseases whose incidence increases with age. This review focuses on the role of AICD and DICD dysfunction in the ageing process and highlights emerging anti-ageing therapeutical strategies offered by apoptosis re-modulation. The challenge for the future is to identify factors and signals that regulate apoptotic processes and determine if selective apoptosis manipulation in specific lymphocyte subsets could preserve immune function in the elderly, contributing to successful ageing.

Adhesion molecules on peripheral blood lymphocyte subpopulations in the elderly.

Adhesion molecules, such as CD49d, CD50 and CD62L, have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations are present in aged subjects, we studied, by means of triple colour whole blood immunostaining and multiparametric flow cytometry, the expression and intensity level (MFI) of these molecules on peripheral blood lymphocyte subpopulations from 23 healthy elderly subjects and 13 young controls. In the elderly a decrease in total peripheral blood lymphocytes bearing CD62L antigen was observed (39 +/- 13% vs 63 +/- 6% and 745 +/- 312/mm3 vs 1,393 +/- 407/mm3; p<0.001), whereas the numbers of lymphocytes expressing CD49d and CD50 antigens were comparable in aged and young subjects. In addition, CD50 and CD62L MFI values on total peripheral blood lymphocytes were higher in elderly than in young subjects (5.23 +/- 1.03 vs 4.18 +/- 0.44, p = 0.001 and 2.60 +/- 0.35 vs 2.21 +/- 0.40, p = 0.005 respectively) while the intensity expression of CD49d was unchanged. The percentages and absolute numbers of T and B lymphocytes expressing CD62L were decreased in elderly compared to young subjects (CD62L+CD3+: 43 +/- 15% vs 66 +/- 9% and 581 +/- 257/mm3 vs 1,028 +/- 418/mm3, p<0.001; CD62L+CD19+: 78 +/- 12% vs 90 +/- 4%, p < 0.005 and 103 +/- 64/mm3 vs 207 +/- 98, p < 0.001). A decrease in the proportion of CD62L bearing NK cells was also observed in the elderly (25 +/- 14% vs 46 +/- 24%, p<0.005), although their absolute number was unchanged. No significant differences were detected in the proportion of T, B and NK lymphocytes expressing CD49d and CD50 antigens and only the absolute numbers of B cells expressing these adhesion molecules were lower in elderly (CD49d+CD19+: 121 +/- 71/mm3 and CD50+CD19+: 107 +/- 73/mm3) compared to young donors (CD49d+CD19+: 248 +/- 112/mm3 and CD50+CD19+: 235 +/- 120/mm3, p < 0.001). Moreover, the intensity of adhesion molecule expression was differentially modulated in the elderly depending on the specific lymphocyte cell population considered. The densities of CD49d, CD50 and CD62L antigens on B and NK lymphocytes from the two age groups were not different; on the contrary, T lymphocytes from elderly donors exhibited increased CD49d (1.69 +/- 0.09 vs 1.62 +/- 0.07, p < 0.05), CD50 (4.98 +/- 1.16 vs 3.77 +/- 0.46, p < 0.001) and CD62L (2.26 +/- 0.38 vs 1.99 +/- 0.37, p < 0.05) MFI values compared to young donors.

7-Ketocholesterol and 5,6-Secosterol Modulate Differently the Stress-Activated Mitogen-Activated Protein Kinases (MAPKs) in Liver Cells

Enhanced oxidative stress is a common feature of liver diseases and contributes to chronic liver disease (CLD) progression by inducing fibrogenesis during liver regeneration. Peroxidation products of cholesterol metabolism, named oxysterols, are new and reliable markers of oxidative stress in vivo. Patients affected by CLDs present high plasma levels of oxysterols, raising the question of the origin and biological relevance of these compounds in the pathophysiology of chronic liver damage. The aim of this study was to examine the molecular basis of the biological effects of oxysterols on liver‐derived cells, HepG2 and Huh7. Cells were treated with different concentrations (10−9 to 10−5 M) of 7‐ketocholesterol used as a reference, and 5,6‐secosterol, a recently discovered oxysterol. FACS investigations, caspase‐3 activation, and Sytox Green immunofluorescent assay showed that pathological concentrations of oxysterols induced necrosis (30–50%) after 48 h of treatment. The two analyzed compounds displayed a similar, but not identical, behavior. In fact, 5,6‐secosterol, but not 7‐ketocholesterol, induced cell senescence. Notably, low concentrations of 5,6‐secosterol caused a sustained activation of ERK1/2, inducing cell proliferation, this unexpected behavior should be better characterized by further studies. Since enhanced oxidative stress is known to worsen liver chronic hepatitis and frequently results in overall decreased cellular survival, our data suggest the important and different role oxysterols may have in interfering with physiological liver tissue regeneration in injured human liver. Antioxidant treatment may provide a highly specific and effective mean to counteract the common consequences of oxidative stress on chronic hepatitis, such as fibrosis/cirrhosis and liver failure. J. Cell. Physiol. 222: 586–595, 2010.