Immunology of Aging: the Birth of Inflammaging.

The inflammaging concept was introduced in 2000 by Prof. Franceschi. This was an evolutionary or rather a revolutionary conceptualization of the immune changes in response to a lifelong stress. This conceptualization permitted to consider the lifelong proinflammatory process as an adaptation which could eventually lead to either beneficial or detrimental consequences. This dichotomy is influenced by both the genetics and the environment. Depending on which way prevails in an individual, the outcome may be healthy longevity or pathological aging burdened with aging-related diseases. The concept of inflammaging has also revealed the complex, systemic nature of aging. Thus, this conceptualization opens the way to consider age-related processes in their complexity, meaning that not only the process but also all counter-processes should be considered. It has also opened the way to add new concepts to the original one, leading to better understanding of the nature of inflammaging and of aging itself. Finally, it showed the way towards potential multimodal interventions involving a holistic approach to optimize the aging process towards a healthy longevity.

Immunosenescence is both functional/adaptive and dysfunctional/maladaptive.

Alterations in the immune system with aging are considered to underlie many age-related diseases. However, many elderly individuals remain healthy until even a very advanced age. There is also an increase in numbers of centenarians and their apparent fitness. We should therefore change our unilaterally detrimental consideration of age-related immune changes. Recent data taking into consideration the immunobiography concept may allow for meaningful distinctions among various aging trajectories. This implies that the aging immune system has a homeodynamic characteristic balanced between adaptive and maladaptive aspects. The survival and health of an individual depends from the equilibrium of this balance. In this article, we highlight which parts of the aging of the immune system may be considered adaptive in contrast to those that may be maladaptive.

Proliferation rate and penicillin binding of enterococcal isolates from patients with immunosupression.

Low proliferation rate of bacterial populations was recently assumed to be a reason for higher resistance to antibiotics and appearance of many chronic infections. Slowly growing populations, called 'small colony variants' (SCVs) have been described in many bacterial species to make from as low as 0·02% up to 46% of population. Thirty enterococcal strains from urine and faeces of renal transplant recipients with asymptomatic, insignificant bacteriuria were studied. Growth characteristics were estimated by microculture and OD reading after 1, 3 and 5 h of culture. At the same time, penicillin binding and changes of aggregation of the cells were analysed by flow cytometry. The results of our study showed high diversity of the proliferation rates among studied isolates. Based on proliferation rates and aggregation, six of studied strains (20%) could be considered as SCVs-like. Significantly lower binding of penicillin was also observed for these SCV-like strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides relevant information about prevalence of enterococcal strains with low proliferation rate (likely small colony variant (SCV)) among kidney transplant recipients. Percentage of such strains in this cohort was relatively high (20%). Additionally, penicillin binding of these strains measured even at the beginning of proliferation (after 1 and 3 h of incubation), was significantly lower than among other strains. Finally, all of them were determined as penicillin resistant, with minimal inhibitory concentration value above 256 µg ml-1 . As the risk of systemic infections caused by such strains is probably higher than in case of other strains, screening for the SCVs in this group of patients should be recommended.

Intracellular signalling pathways: targets to reverse immunosenescence.

Ageing is a very complex process, the result of the dysregulation of multiple systems interacting in many ways. A prominent change occurring with ageing is related to the architecture and functioning of the immune system, viewed commonly as detrimental and termed 'immunosenescence'. However, age-associated changes may also lead to increased function in certain respects, which can be viewed as adaptive. None the less, on balance it is well-recognized that immunosenescence is accompanied by the low-grade inflammation observed commonly in elderly people, which has been dubbed 'inflamm-ageing'. The exact cause and significance of all these changes is not clear, but there is a consensus that they are related to the occurrence of chronic non-infectious age-associated disease, as well as increased susceptibility to infections. Alterations to immune cell signalling may be a prominent cause of malfunctioning immunity. Emerging attempts to reverse immunosenescence have recently targeted the signalling pathways in various different cell types of the immune system. Here, we review and discuss alterations in the signalling pathways of immune cells with ageing and consider current targets and means to modulate altered functions. We discuss the potential dangers as well as the benefits of these interventions, and consider future approaches to this problem.

From inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation.

Aging is accompanied by many physiological changes including those in the immune system. These changes are designated as immunosenescence indicating that age induces a decrease in immune functions. However, since many years we know that some aspects are not decreasing but instead are increasing like the pro-inflammatory activity by the innate immune cells, especially by monocytes/macrophages. Recently it became evident that these cells may possess a sort of memory called trained memory sustained by epigenetic changes occurring long after even in the absence of the initiator aggressor. In this review we are reviewing evidences that such changes may occur in aging and describe the relationship between inflamm-aging and immunosenescence as an adaptation/remodelling process leading on one hand to increased inflammation and on the other to decreased immune response (immune-paralysis) mastered by the innate immune system. These changes may collectively induce a state of alertness which assure an immune response even if ultimately resulting in age-related deleterious inflammatory diseases.

Older age of rheumatoid arthritis onset is associated with higher activation status of peripheral blood CD4(+) T cells and disease activity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, with a clinical manifestation both systemic and in joints. It has been suggested that age at disease onset and/or patients' age have influence on disease activity and clinical outcome. The reasons for the different course of RA in older people are not known; however, the activation status of peripheral blood lymphocytes could be responsible. Our aim was to relate expression of activation markers in peripheral blood CD4(+) T cells of RA patients with patients' age and/or onset age and disease activity measured by DAS28. Seventy RA patients were included into the immunological study. Two separation criteria were performed: based on age of RA onset and on the biological age of patients. We examined different activation markers, CD69, CD25, CD95 and human leucocyte antigen D-related (HLA-DR), on the CD4(+) T cell surface. Division of RA patients in 10-year intervals at 40, 50 and 60 years revealed that RA patients with later disease onset were characterized by higher DAS28. This phenomenon was not limited to the division at 60 years of age but, surprisingly, the major differences were found for the 40-year onset division. Analysis of all four components of DAS28 revealed that disease activity in older disease onset was dependent on all components. Older-onset RA patients had a higher percentage of CD4(+) CD25(+) and CD4(+) CD95(+) T cells. Summarizing the major differences in DAS28 and activation status of CD4(+) T cells observed for onset of disease at 40 years seems to be the most informative about the immunological status of RA patients.

Aging, frailty and age-related diseases.

The concept of frailty as a medically distinct syndrome has evolved based on the clinical experience of geriatricians and is clinically well recognizable. Frailty is a nonspecific state of vulnerability, which reflects multisystem physiological change. These changes underlying frailty do not always achieve disease status, so some people, usually very elderly, are frail without a specific life threatening illness. Current thinking is that not only physical but also psychological, cognitive and social factors contribute to this syndrome and need to be taken into account in its definition and treatment. Together, these signs and symptoms seem to reflect a reduced functional reserve and consequent decrease in adaptation (resilience) to any sort of stressor and perhaps even in the absence of extrinsic stressors. The overall consequence is that frail elderly are at higher risk for accelerated physical and cognitive decline, disability and death. All these characteristics associated with frailty can easily be applied to the definition and characterization of the aging process per se and there is little consensus in the literature concerning the physiological/biological pathways associated with or determining frailty. It is probably true to say that a consensus view would implicate heightened chronic systemic inflammation as a major contributor to frailty. This review will focus on the relationship between aging, frailty and age-related diseases, and will highlight possible interventions to reduce the occurrence and effects of frailty in elderly people.

Different distribution of CD4 and CD8 T cells in synovial membrane and peripheral blood of rheumatoid arthritis and osteoarthritis patients.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are chronic diseases associated with morphological joint changes. Synovial membrane (SM) involvement was established for RA, but the data for OA are limited, because OA is usually regarded as noninflammatory disease. Changes in immune system in RA are not limited to joints, and the significant role of T cells of peripheral blood (PB) is not disputable. However, there is still an open debate about PB immunological profile in OA. Therefore, we decided to measure the distribution of CD4+ and CD8+ T cells, regarding CD28 expression, both in PB and SM of RA and OA patients, on the same day. Altogether, eleven RA patients, 11 OA patients and similar numbers of age-matched healthy controls were included into the study. Flow cytometry was used for T cells subpopulation distinguishing and quantification; monoclonal antibodies against CD3, CD4, CD8 and CD28 with different fluorochromes were used for stainings. The RA patients had significantly higher percentage of CD3+4+ cells in PB as compared to OA patients and relevant control group. Both within the CD4+ and CD8+ compartments, significantly lower percentages of cells bearing the CD28 marker were found in the PB of OA as compared to RA patients. The proportion of CD3+CD4+ cells in SM was dependent on age of OA patients, older OA patients had significantly higher value of their SM/blood ratio than RA patients. Older OA subjects were also characterized by higher values of the SM/blood ratio of both CD4+CD28+ and CD8+CD28+ subpopulations than RA or younger OA patients. In conclusion, in contrast to the traditional view of OA disease, our results give support to the hypothesis that OA may also (like RA) be a disease with a local immunological involvement.

Cytoprotective effect of lithium against spontaneous and induced apoptosis of lymphoid cell line MOLT-4.

Lithium (Li) is still useful in the treatment of bipolar disorder. Cellular mechanisms of Li action are not fully understood and include some cytoprotective properties. Data concerning Li effect on the apoptotic mechanisms in cells other than neurons are fragmentary and contradictory. We have investigated anti-apoptotic activity of Li in a lymphoid derived MOLT-4 cell line. Spontaneous and camptothecin-induced apoptosis was analyzed in cells treated with 0-20 mM Li carbonate. Early apoptosis was identified as significant mitochondrial depolarization (JC-1 staining). Later stages of apoptosis were estimated with annexin V binding and by the proportion of cells containing sub-G1 amounts of DNA (PI staining). We have observed a biphasic effect of Li on the proportion of spontaneously apoptotic cells;namely, low (therapeutic) concentrations of Li had a significant effect stabilizing the mitochondrial membrane polarization, while 10 and 20mM Li increased apoptosis. The latter could be seen both as mitochondrial depolarization as well as an increased proportion of sub-G1 cells, accompanied by reduced proportion of S phase cells. Li at concentrations above 2 mM had a significant, dose-dependent, anti-apoptotic effect on the cells undergoing camptothecin induced apoptosis. In conclusion, demonstrated cytoprotective effect of Li is at least partially related to stabilization of mitochondrial membrane potential and to the reduction of DNA damaging effects in proliferating cells; both may form part of the mechanism through which Li is useful in therapy of bipolar disorder, but may have more general consequences.

Age-related increase of frequency of a new, phenotypically distinct subpopulation of human peripheral blood T cells expressing lowered levels of CD4.

Aging is associated with modifications of T-cell phenotype and function, leading to impaired activation in response to both new and recall antigens. It is not known if T-cell activation results in elimination of a number of the CD4 molecules from the cell surface, as is the case with CD3/T-cell receptor complexes, or how aging influences the process. The T cells of young and elderly donors with reduced expression of CD4 were examined to see whether these cells exhibit other phenotypic features suggesting their active state. It was found that T lymphocytes expressing CD4 can be divided into 2 semidiscrete subpopulations: the major (CD4(+)) population, in which the level of expression of CD4 is constant and high, and a minor population (CD4(lo)), in which the expression of CD4 can be up to an order of magnitude lower than on the CD4(+) cells. The proportion of CD4(lo) cells is age dependent and highly variable in the apparently healthy human population, with the expression of CD4 ranging from around 10% of all peripheral blood lymphocytes in the young to more than 30% in the elderly. Lowered expression of CD4 is correlated with a reduced expression of CD3, as well as with a decreased amount of CD28 and CD95Fas. Activation of CD4(lo) cells is suggested by their expression of CD25 and increased amounts of HLA-DR. Phenotypic characteristics of the CD4(lo) T-cell subpopulation suggest that it might be formed by (perhaps chronically) activated, temporarily apoptosis-resistant cells, possibly accumulating in the elderly. (Blood. 2001;98:1100-1107)

Expression and activity of P-glycoprotein in transplantable hamster melanomas.

In the study described here we investigated the possibility of an association between the aggressiveness of melanoma and multidrug resistance phenotype by analyzing the expression and activity of P-glycoprotein (Pgp) in two genetically related transplantable hamster melanomas--a melanotic (Ma) and an amelanotic (Ab) form --which differed in aggressiveness and metastatic potential. Flow cytometric analysis of Pgp activity (using a verapamil-sensitive rhodamine R123 exclusion test) as well as Western blotting of cellular lysates showed its preferential (although not very marked) expression in the Ab melanoma cells. The Ab melanoma cells also exhibited a higher proportion of tumor-infiltrating lymphocytes (TIL), mostly of T cell phenotype, that may have reflected a higher immunogenicity of the tumor. In conclusion, Pgp activity appeared to be associated with less-differentiated more aggressively metastasizing melanoma (the Ab variant) although its role in maintaining this phenotype remains to be established.

Flow cytometric estimation of the plasma membrane diversity of transplantable melanomas, using annexin V.

Using a recently described flow cytometric assay probing for cell surface exposure of phosphatidylserine with fluoresceine-labeled annexin V, we attempted to establish if there existed any differences in the phospholipid bilayer of the plasma membranes of melanoma cells isolated from two lines of a hamster transplantable melanoma characterized by a common origin but differing in many biological features. In contrast to control nonstaining cells, the cells of both melanoma lines bound annexin V, but at a different rate: 88% of melanotic and 94% of amelanotic melanoma cells were annexin V positive. Among cells of the native melanotic melanoma line we distinguished only one cell population binding annexin but in some experiments with the amelanotic melanoma we observed two annexin V positive cell populations with a different fluorescence intensity. It is possible that these differences in annexin V binding to melanoma cell membranes reflect some changes in the phospholipid bilayer, associated with the progression of these tumors.

Diversity of the plasma membrane properties of transplantable hamster melanomas with regard to the expression of P-glycoprotein.

A comparison of the expression of P-glycoprotein (Pgp) was performed in two forms of hamster transplantable melanomas of common origin, but differing in growth rates and levels of differentiation. The expression of P-glycoprotein in plasma membranes of these two forms of melanomas was estimated by the western blot analysis and the transport activity of the Pgp compared by flow cytometry. It was observed that a spontaneous alteration in the original melanotic melanoma leading to a formation of the amelanotic form characterized by higher growth rate, greater anaplasticity and leading to the animals' death after a shorter time from inoculation, was accompanied by a decrease in the Pgp expression and activity, due to simultaneous appearance of a small population of amelanotic cells with high Pgp expression and activity, and disappearance of this activity from the major population. It is possible, that the activity of Pgp in the melanoma cell membranes reflects the degree of cell differentiation.

[Impaired signal transduction in T-lymphocytes of the aged]. / Zaburzenia transdukcji sygnalu towarzyszace starzeniu sie limfocytów T.

An age-related dysfunction of the immune system, and especially of the T lymphocytes, is the most common feature observed during aging. It is well recognized by now that changes in the molecular mechanisms connecting the antigen receptor of the T cell with its nuclear machinery, commonly called 'signal transduction pathways' are the basis for this dysfunction. This paper is an up-to-date review of current literature of the problem, describing age-related changes in the functioning of three major, complementary pathways of signal transduction in murine and human T cell: IP3/Ca2+/calcineurin, DAG/protein kinase C (PKC) and Ras/MAP kinases, discovered so far.

Calcium signal abnormalities in murine T lymphocytes that express the multidrug transporter P-glycoprotein.

Previous work has established that aging in mice leads to an accumulation of T cells that express high levels of P-glycoprotein, a plasma membrane pump that mediates multiple drug resistance in tumor cells but whose function in normal T cells is still obscure. Pgp+ cells seem to be functionally defective: isolated from the CD4 memory population of young mice, they are unresponsive to T cell receptor-dependent stimuli in tests for proliferation and cytokine production. The proliferative defect can, however, be overcome by exposure to PMA plus the calcium ionophore ionomycin, suggesting that the Pgp+ cells may have a specific defect in calcium signal generation. We show here that Pgp+ T cells, from young or old mice, do indeed show smaller changes in intracellular calcium ion concentration than Pgp- cells, when activated either by Con A, anti-CD3 antibodies, or ionomycin. The difference between Pgp+ and Pgp- cells is apparent even in experiments on isolated CD4 memory T cells from young mice and thus is not simply a consequence of the age-dependent increase in memory cell numbers. Although the molecular basis for the abnormality in calcium signal generation by Pgp+ cells is still uncertain, our data suggest that the effect could be due to inter-subset differences in levels of sorcin, a 22 kDa cytoplasmic protein that is co-expressed with P-glycoprotein in many tumor cells and which binds free calcium ion with high affinity. Sorcin levels are higher in Pgp+ CD4 cells than in Pgp- CD4 cells of young mice and increase with age in CD4 cells, consistent with the hypothesis that sorcin interferes with calcium signals in the age-sensitive Pgp+ T cell subset.

Early activation defects in T lymphocytes from aged mice.

Aging affects both calcium signals and protein kinase cascades in mouse T lymphocytes. The decline in calcium signal development largely represents differences between naive and memory T cells; the latter are resistant to increases in calcium concentration, and are more common in aged mice. Aging leads to declines in phosphorylation of a wide range of substrates in T cells stimulated by either anti-CD3 antibodies or by substances, such as phorbol myristate acetate (PMA) or ionomycin, that act at intracellular sites, but some phosphoproteins respond only in old T cells, and others respond regardless of age. Tyrosine phosphorylation of the CD3 zeta chain declines with age, both in resting T cells and after activation, but the proportion of Zap-70 that is bound to CD3 zeta increases in T cells from old mice. Zap-70 function and phosphorylation of CD3 zeta-associated Zap-70 change only slightly after stimulation of T cells by anti-CD3 and anti-CD4, and are at similar levels in activated old and young T cells. Nonetheless, induction of Raf-1, MEK, and ERK kinase activity declines with age in CD4 T cells. The effect of aging on T-cell activation is not simply an overall decline in signal intensity, but a set of qualitative changes that differ among subsets and depend at least partly on the nature of the stimulus.

Reciprocal expression of P-glycoprotein and TAP1 accompanied by higher expression of MHC class I antigens in T cells of old mice.

We have shown previously that (a) aging leads to an increase in the proportion of murine splenic T cells that express high activity of P-glycoprotein (PGP), the ATP-dependent plasma membrane pump that mediates multiple drug resistance, and (b) PGPhi CD4 memory cells from mice of any age do not proliferate or secrete IL-4 after activation with anti-CD3 and IL2. We now report that the age-associated increase in expression of MHC Class I molecules is limited to the subset of T cells that overexpress PGP and thus extrude the fluorochrome R123 (the "R123lo" subset). Although H-2 levels increase on T cells of old mice, the levels of TAP1, a component of the polypeptide pump responsible for assembly and internal transport of Class I MHC molecules, decline, unexpectedly, by about fourfold in T cells from old donors. Thus, aging leads to reciprocal changes in the level of T-cell expression of PGP and TAP1, two closely related members of the ABC superfamily of peptide transport proteins.

Changes of natural killer cytotoxic activity and natural killer sensitivity during growth of Bomirski melanotic (Ma) and amelanotic (Ab) melanomas.

Natural killer (NK) cytotoxic activity during the growth of melanotic (Ma) and amelanotic (Ab) variants of Bomirski hamster melanoma, in the blood and the spleen, was examined. The melanoma variants differed in their growth rate and metastatic pattern. Ability to form conjugates by effectors with targets and cytotoxic effects were compared. As targets K562 cells and tumor cells were used. It was found that during the growth of Ma melanoma activity of NK cells was not changing but the sensitivity of tumor cells was decreasing. However, during growth of Ab melanoma both NK activity and NK sensitivity were reduced.

Extrusion of the P glycoprotein substrate rhodamine-123 distinguishes CD4 memory T cell subsets that differ in IL-2-driven IL-4 production.

Approximately 25% of T cells in young mice are able to extrude the fluorescent P glycoprotein substrate Rhodamine-123 (R123), and these R123low cells are present within the naive and memory cell populations of both CD4 and CD8 type. The proportion of R123low T cells increases with age to approximately 60% in mice older than 18 mo of age. CD4 memory T cells from young mice secrete more IL-4 compared with cells from old donors when activated by anti-CD3 and further cultured for 7 to 10 days in the presence of IL-2. To determine whether this age-related decline in IL-4 production was related to the parallel accumulation of R123low T cells within the CD4 memory subset, we compared IL-4 production in cell preparations enriched for R123high and R123low cells by electronic cell sorting. IL-2-driven IL-4 production by CD3-activated CD4 memory cells was found to be limited almost entirely to the R123high subset, i.e., the subset that declines with age. Proliferation under these culture conditions also declined with age and was also much more vigorous in R123high than in R123low cells. Not all R123low T cells, however, were resistant to IL-2-dependent proliferation and differentiation: limit dilution analyses showed nearly equal proportions of Con A-responsive proliferative and cytotoxic clones within the R123high and R123low subsets of CD8 naive cells. These data show that P glycoprotein-mediated extrusion of R123 delineates CD4 memory T cell subsets that differ in function.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-dependent efflux of K+ ions from stimulated T lymphocytes of young and old mice.

The changes in the rate of efflux of potassium cations from ionomycin-, A23187-, and Concanavalin A (Con A)-stimulated lymphocytes from young and old mice were studied with an ion-selective electrode in a 'half-micro' system. It was found that, for both types of stimuli, the maximal rate of Ca(2+)-dependent efflux of K+ from young T cells was more than twice that attained by old cells. A bimodality of the curve illustrating change of efflux rate was observed, indicating possible existence of two types of Ca(2+)-dependent conductances. The kinetics of change of the efflux rate was similar for young and old cells stimulated with calcium ionophores, but differed in the cells stimulated with Con A. There, a lag period between mitogen addition and onset of measurable K+ efflux was absent in the case of old T cells, suggesting that function of other (Ca-independent?) mechanisms of K+ efflux during the mitogen stimulation may also be changed there. The measured efflux of K+ was only partially dependent on the extracellular Ca2+. Also, it was quantitatively different in a 'physiological' medium containing 140 mM Na+, as compared to a sodium-free medium. Different blockers of potassium, calcium and sodium channels had at least partially inhibitory effect on the measured flux. Presented findings indicate that potassium conductance through Ca(2+)-gated K+ channels is impaired in T cells of old mice. Ca(2+)-dependent efflux of K+ in murine T cells is apparently conducted by a specific class of membrane channels, possibly consisting of two types of channels with different activation kinetics and pharmacological sensitivities (expressed in different subpopulations of T cells?). Impaired potassium conductance in old T cells is discussed as one of possible causes of age-related dysfunction of the immune system.