Granulocyte and natural killer activity in the elderly.

The deterioration of the immune system in ageing, 'immunosenescence', is thought to contribute to increased morbidity and mortality from infections and possibly autoimmune diseases and cancer. The most profound changes involve effector and immunoregulatory T-cell functions. Immunosenescence appears also to be related to changes in non specific immunity as well. In the present study we have assessed superoxide production, chemotaxis and the expression of the apoptosis-related molecule APO1/Fas (CD95) on neutrophils (PMN) from young and old subjects. Furthermore, we have measured the basal natural killer (NK) activity of young and elderly subjects and we have compared the number of CD16+ cells found in these two groups. We observed a significant decrease age-related both of formation of O2- and chemotaxis whereas no significant correlation between age and the expression of CD95 on granulocyte membrane was demonstrated, suggesting that an increase age-related of CD95-linked apoptosis of PMN should be not an important determinant in the decreased PMN function. We also observed a significant correlation between age and NK activity. The decreased NK cell function was not due to a decreased number of NK cells in effector cell preparations since the number of CD16+ cells was significantly increased in old subjects. In conclusion, our results show that in the elderly there is also a deficit of the aspecific immunity that might play a role in the pathogenic mechanisms of the immunosenescence.

HLA-B8,DR3 haplotype affects lymphocyte blood levels.

The number of lymphocytes in the blood is constant, pointing to an effective control of circulating lymphocyte values. The mechanisms of this regulation are uncertain, although it is likely that the number of blood lymphocytes is conditioned by hormones, homing factors and cytokines whose production is at least partly restrained by genetic factors. Particularly genetic factors linked to major histocompatibility complex (MHC) appear to be involved. In human beings a decreased number of blood lymphocytes has been described in healthy subjects carrying the Human Leucocyte Antigens (HLA) haplotype HLA-B8,DR3. In the present study, to inquire into the mechanisms of this lymphocyte decreased number, we have performed an analysis of blood subset values in these subjects. When the absolute values of lymphocytes were analysed according to HLA phenotype, HLA-B8,DR3 positive subjects (N = 26) displayed significantly lower values as compared to HLA-B8,DR3 negative ones (N = 282). The analysis of lymphocyte subpopulations performed by flow cytometry in 72 subjects did not show significant changes in lymphocyte subset percentages between HLA-B8,DR3 positive subjects and negative ones. Thus, the decrease of circulating lymphocytes seems to be due to a reduction of cell number affecting all lymphocyte subsets rather than a single cell subpopulation. The analysis of in vitro spontaneous apoptosis performed by flow cytometry in a smaller sample of subjects showed a significant increase of spontaneous apoptosis in lymphocytes from HLA-B8,DR3 positive individuals suggesting a possible explanation for the deviation from normal lymphocyte count observed in these subjects. However it is intriguing that a decreased number of blood lymphocytes can be observed in healthy HLA-B8,DR3 positive subjects but also in autoimmune diseases linked to this haplotype like systemic lupus erythematosus and insulin-dependent diabetes. Furthermore, in our opinion, this finding is to be kept in mind in evaluating hematological parameters in healthy subjects.

In vitro cytokine production by HLA-B8,DR3 positive subjects.

It is well known that healthy subjects carrying the HLA-B8,DR3 haplotype may show an impairment of immune system, the T cells being the most affected. To gain insight into the mechanism(s) of the impairment displayed by these subjects, efforts have been centered on the study of in vitro cytokine production because of the pivotal role played by these mediators in the activation and control of several immune functions. The available results indicate that the ability to several immune functions. The available results indicate that the ability to produce interleukin-1 (IL-1), IL-2 and the soluble form of its receptor (sIL-2R) is impaired in HLA-B8,DR3 positive healthy subjects. To better characterize the cytokine production capacity of HLA-B8,DR3 positive subjects, we have investigated the pattern of in vitro production of IL-2, sIL-2R, IL-4. IL-6 and gamma-interferon (gamma-IFN) by mononuclear cells from HLA-B8, DR3 positive subjects after phytohaemoagglutinin stimulation. A significant decrease of IL-2, sIL-2R and gamma-IFN production by HLA-B8,DR3 positive subjects was observed. No significant difference was instead found between the HLA-B8,DR3 positive subjects and the negative ones as regards IL-4 and IL-6 production. We suggest that this imbalanced cytokine production may well account for the pattern of immune response that may observed in HLA-B8,DR3 positive subjects, i.e. a normal or increased humoral response in face of a low T cell immune responsiveness.

Natural killer and lymphokine-activated killer activity in HLA-B8,DR3-positive subjects.

The haplotype HLA-B8,DR3 is over-represented in several autoimmune diseases, implying that genes predisposing people to these disorders are linked to this haplotype. In these diseases, various dysfunctions reflecting an impairment of the immune system have been found. Several reports indicate also that in HLA-B8,DR3-positive healthy subjects similar disorders may be demonstrated. In the present work, we have evaluated NK and LAK activity in these subjects. The study has been performed on monocyte-depleted peripheral blood MNCs by using, the K-562 cell line as a target for NK activity and the HL-60 cell line for as a target LAK activity. LAK cells were obtained by incubating MNCs for 3 days with 100 U/ml of rIL-2. The results of our experiments demonstrate that NK cell activity is significantly decreased in healthy subjects bearing the HLA-B8,DR3 haplotype. Since the number of circulating CD16+ cells is not significantly different between HLA-B8,DR3-positive subjects and negative ones, it is unlikely that this defect is due to a decreased number of NK cells in effector cell preparations. The observation that the treatment with rIL-2 can restore the killer activity of MNCs from these subjects suggests instead that the reduced NK activity may be due at least in part to the imbalance of cytokine network that has been demonstrated in HLA-B8,DR3-positive subjects. Finally, since a decreased NK activity has been reported in the autoimmune diseases linked to this haplotype, our results support the suggestion that immunologic changes observed in autoimmune diseases reflect systemic regulatory disorders that have a genetic basis.

HLA-B8,DR3 phenotype and the antibody response against Epstein-Barr virus.

Antibodies against the viral capsid antigen (VCA) and nuclear antigens (EBNAs) of the Epstein-Barr virus (EBV) were determined in a sample of Sicilian population. A significant correlation was observed between HLA-B8,DR3 phenotype and reduced titres of antibodies to EBNAs, whereas HLA-B8,DR3 positive individuals displayed levels of antibodies to VCA comparable to those of HLA-B8,DR3 negative ones. These results further strengthen the suggestion that HLA-B8,DR3 positive subjects are low responders and that the depth of immune response depends on the fashion of antigenic challenge.

Biological significance of soluble IL-2 receptor.

A NUMBER of receptors for growth factors and differentiation antigens have been found to be secreted or released by cells. Following mononuclear cell (MNC) activation and interleukin-2 receptor (IL-2R) expression, a soluble form of the Alpha;-chain of IL-2R (sIL-2R) is released. The sIL-2R has been shown to be present in the culture supernatants of activated MNCs as well as in normal sera and, in higher amounts, in sera from subjects affected by several diseases including neoplastic, infectious and autoimmune ones, and in sera from transplanted patients suffering allograft rejection. The blood sIL-2R levels depend on the number of producing cells and the number of molecules per cell, so that sIL-2R blood values may represent an index of the number and the functional state of producing cells, both normal and neoplastic. Thus, monitoring of the immune system, mostly T-cells and haematological malignancies might be targets for the measurement of sIL-2R. Since many conditions may influence sIL-2R production, little diagnostic use may result from these measurements. However, since blood sIL-2R levels may correlate with disease progression and/or response to therapy, their measurement may be a useful index of activity and extent of disease. The precise biological role of the soluble form of the IL-2R is still a matter of debate. However, we know that increased sIL-2R levels may be observed in association with several immunological abnormalities and that sIL-2R is able to bind IL-2. It is conceivable then that in these conditions the excess sIL-2R released in vivo by activated lymphoid cells or by neoplastic cells may somehow regulate IL-2-dependent processes. On the other hand, it cannot exclude that sIL-2R is a by-product without biological significance. Finally, it is puzzling that in many conditions in which an increase of blood sIL-2R values has been observed, MNCs display a decreased in vitro capacity to produce sIL-2R. These seemingly contrasting findings are discussed in the light of the data showing that sIL-2R production correlates with IL-2 production.

gamma-Interferon, interleukin-4 and interleukin-6 in vitro production in old subjects.

It is well known that ageing is associated with various alterations of the lymphoid cell functions. Although both B and T cell are affected, the last appear to be more sensitive to ageing process. During the past years, to gain insight into thé mechanism(s) of this impairment, effort has been centered on the helper T cells specifically engaged in the production of interleukin-2 (IL-2) because of the pivotal role played by this cytokine in the activation of several immune functions. The results have demonstrated that the ability to produce IL-2 declines with age. In this paper we report the results of a study performed to determine the influence of age on the capacity to produce gamma-interferon (gamma-IFN), interleukin-4 (IL-4) and interleukin-6 (IL-6). Mononuclear cells from young and old subjects were assessed for cytokine producing capacity in response to phytohaemagglutinin stimulation. A significant decrease of gamma-IFN production by old subjects has been observed. No significant difference was instead observed between the old subjects and the young ones as regards IL-4 and IL-6 production. We suggest that this imbalanced cytokine production may well account for the pattern of immune response which may be observed in elderly, i.e. a normal or increased humoral response in face of a low T cell immune responsiveness.

The effect of age on mitogen responsive T cell precursors in human beings is completely restored by interleukin-2.

It is well known that the function of T lymphocytes is significantly impaired by advancing age. In the present study, attempts have been made to further characterize the T cell impairment of elderly subjects. Thus, we have performed limiting dilution microculture analysis to evaluate the precursor frequency of T lymphocytes responding to a mitogenic stimulus in old and young subjects. Furthermore we have evaluated the activity of recombinant interleukin-2 (rIL-2) on these cells. The results demonstrate that in older subjects the frequency of these precursors is significantly decreased. The in vitro treatment with rIL-2 increased the frequency of mitogen responsive T lymphocyte precursors in both groups so that the difference between the two groups was not significant. Thus present results extend the findings demonstrating that older subjects display an impairment of T cell functions and that IL-2 treatment may correct these alterations. In particular, they confirm the hypothesis that age-associated functional changes are more likely due to diminished numbers of reactive cells, than to a decline in the activity of all cells.

IgD serum levels are influenced by HLA-DR phenotype.

In the present paper we have evaluated IgD serum levels of 84 randomly selected HLA-typed healthy Sicilians. The values were analysed according to age, sex and HLA-DR phenotypes. No correlation between age and IgD serum levels was found in our population since all subjects were in a narrow age range. Furthermore, no significant association was found between IgD serum levels and gender of studied subjects. The evaluation of IgD serum levels according to HLA-DR phenotypes revealed that HLA-DR1 positive subjects displayed significantly higher values. These results are in agreement with previous reports showing that HLA phenotypes may be involved in the control of serum immunoglobulin levels. Furthermore, present data strengthen our suggestion that HLA-DR1 phenotype is related to the 'high responder' immunological profile.

Blood antiphospholipid antibody levels are influenced by age, sex and HLA-B8,DR3 phenotype.

Antiphospholipid antibodies (APA) are known to be associated with a number of seemingly heterogeneous pathological conditions that are part of the antiphospholipid syndrome, formerly called anticardiolipin syndrome. Recent studies on the mechanism of action of these autoantibodies suggest that we are dealing with a new autoimmune syndrome which may occur either in a primary form or in the context of other autoimmune diseases, mainly systemic lupus erythematosus (SLE). Moreover, increased levels of APA have been found in elderly subjects, who are known to display increased frequency of autoimmune phenomena. It is well known that many autoimmune diseases, including SLE, are associated with HLA antigens, particularly with HLA-B8,DR3 phenotype. In our study, APA serum levels were analyzed in 26 old subjects and in 56 young ones. The results demonstrate that HLA-B8,DR3-positive young females display significantly higher levels of APA than HLA-B8,DR3-negative ones. Interestingly, the same is true for elderly subjects on the whole with respect to young individuals. These data are consistent with previous findings demonstrating that HLA-B8,DR3-positive subjects (mainly female) as well as old subjects display (also in the absence of any clinical manifestation), multiple immune dysfunctions that may underlie the predisposition to autoimmunity.

HLA-B8,DR3 T cell impairment is completely restored by in vitro treatment with interleukin-2.

The activity of recombinant interleukin-2 (rIL-2) on the in vitro lymphocyte proliferative response to phytohemagglutinin mitogen was investigated in healthy HLA-B8,DR3 positive and negative subjects. The response to mitogen, significantly decreased in HLA-B8,DR3 positive subjects, was completely restored by adding rIL-2. Moreover, in HLA-B8,DR3 positive subjects the in vitro treatment with rIL-2 significantly increased the reduced frequency of mitogen responsive T lymphocyte precursors, as assessed by limiting dilution analysis. These data suggest that a decrease in the size of the pool of T cell precursors able to produce IL-2 is responsible for the impairment of T cell function observed in HLA-B8,DR3 positive subjects. Since in autoimmune diseases it is possible to show the same impairment(s) of T cell functions which can be observed in HLA-B8,DR3 positive subjects, these results could be of practical value for the understanding of pathogenetic mechanism(s) of autoimmune diseases and, in case, for therapeutical purposes.