Francesco Gervasi

A genetically determined high setting of TNF-α influences immunologic parameters of HLA-B8,DR3 positive subjects: implications for autoimmunity

The 8.1 ancestral haplotype (AH) is a common Caucasoid haplotype carried by most people who type for HLA-B8,DR3. It seems unique in its association with a wide range of immunopathologic diseases. Healthy subjects bearing this haplotype demonstrate several alterations of immune response. This article will focus on the identification of the mechanism(s) of disease susceptibility of 8.1 AH. In 13 carriers of 8.1 AH, and 43 negative patients, enzyme immune assays serum levels of tumor necrosis factor (TNF)-alpha, soluble endothelial leukocyte adhesion molecule-1 (sELAM-1), cortisol, and interleukin(IL)-10 were determined. In addition, quantification of cytokine produced in vitro after mitogen stimulation was studied, and all subjects were genotyped for alleles at -592, -819, and -1082 nucleotides of IL-10 gene 5 flanking region, which is known to control IL-10 production. Results revealed that 8.1 AH is associated with a high in vivo and in vitro production of TNF-alpha, which in turn seems responsible for increased serum levels of sELAM-1, cortisol, and IL-10. On the contrary, in vitro production of IL-10 is not increased in these patients and there are no differences in allele promoter frequencies between the two groups that might explain the differences in IL-10 serum values. Thus, serum values seem to be the result of the effects of increased serum levels of TNF-alpha and cortisol. In conclusion, the increased spontaneous release of TNF-alpha, which modifies a certain number of immunologic parameters, may be the most characterizing feature of 8.1 AH. The consequent modification of the immunologic scenario might be involved in the predisposition to the impressive number of diseases and the changes in immune response observed in the patients studied.

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.

Apoptosis and ageing

Stimulation of T cells from aged individuals leads to different kinds and/or size of responses if compared with the responses of T cells obtained from young individuals. In fact elderly is associated with a progressive decline of immune response besides an increasing incidence of autoimmune phenomena. These differences might be the result of modified cellular mechanisms controlling the immune system in the course of ageing. The apoptotic deletion of activated T cells has been proposed as the key mechanism to maintain T cell homeostasis, and in this respect CD95 (Fas antigen) seems to play a major role in this course of events. In this study we show that just collected lymphocytes from old subjects displayed an increased expression of the apoptosis molecule CD95. The expression of CD95 and the spontaneous apoptosis showed the same trend. In fact the percentage of apoptotic cells in blood collected from old subjects was enhanced too. The lymphocyte subpopulation analysis by flow cytometry did not show significant changes in T subset percentages between old and young subjects. Moreover mononuclear cells obtained from aged individuals underwent apoptosis in culture in response to a single stimulation with mitogen or anti-CD3, more than mononuclear cells from young controls. To gain insight into mechanisms of this increased apoptosis, experiments were performed to evaluate the behaviors of lymphocytes from old and young donors in respect of interleukin-2 (IL-2) rescue from apoptosis. Results show that IL-2 rescued only a little fraction of cells of old donors from apoptosis when activated by anti-CD3 and that this effect was not related to a different expression of CD95. Thus, during the course of ageing the different regulation of T cell homeostasis might be also explained by the modified proneness of lymphocytes to undergo apoptosis. The contemporaneous demonstration of a reduced Ca2+ influx in lymphoid cells of these subjects allows to suppose that multiple defects play a role in the pathogenesis of immunosenescence.

Measurement of inflammatory mediators of mast cells and eosinophils in native nasal lavage fluid in nasal polyposis.

Background: Nasal polyposis (NP) often coexists with asthma, rhinitis and sinusitis. Polyp histology typically shows chronic, eosinophilic inflammation. The inflammatory cell infiltrate generally includes eosinophils, lymphocytes, plasma cells and mast cells. Objective: To gain insight into the natural history of NP, we analysed mediator levels and leukocyte values in nasal fluids and eosinophil cationic protein (ECP), total IgE levels and eosinophils in the blood in several groups of both allergic and non-allergic patients with nasal polyps and in patients with allergic rhinitis (AR). Methods: Thirty-two patients with nasal polyps entered the study. As a control group, we studied 55 patients with AR, i.e. 20 patients with seasonal AR to grass pollen, 24 with AR sensitive to Parietaria and 11 with AR sensitive to house dust mite (HDM). Eighteen patients with nasal polyps were also allergic patients (8 were sensitive to Parietaria and 10 were sensitive to HDM), whereas 14 were non-allergic patients. Tryptase and histamine values were assayed in nasal fluids, whereas total IgE was determined in serum. ECP values were assayed both in nasal fluids and serum. Eosinophils were quantified both in the blood and nasal fluids. Results: Tryptase levels were significantly higher in the nasal lavages from patients with NP than in those from patients without NP (4.7 vs. 3.5 U/l, p < 0.001) and correlated with symptom scores (rs = 0.42, p < 0.0001). The median levels of histamine in nasal fluids from patients with NP were also significantly higher than those observed in patients without NP (50.0 vs. 21.3 ng/ml, p < 0.001), but did not correlate with symptom scores. Finally, the median levels of ECP in nasal fluids from patients with NP were significantly higher than those observed in patients without NP (38.1 vs. 16.1 ng/ml, p < 0.001) and correlated with symptom scores (rs = 0.38, p < 0.001). Analysis of variance showed that, among the variables studied, the presence of nasal polyps was the factor responsible for the higher levels of tryptase, histamine and ECP in nasal fluids. With regard to leukocyte counts in nasal fluids, no significant differences were observed between rhinitis patients with NP and those without NP. With regard to serum ECP and serum total IgE, no significant differences were detected between the two groups under study. Blood eosinophil levels in patients with NP were significantly higher than those observed in patients without NP (5.8 vs. 5.6, p = 0.002). Analysis of variance showed that both the presence of nasal polyps and the type of sensitisation were important. Considering the total symptom scores, no significant differences were observed between rhinitis patients with NP and those without NP. Conclusions: The present findings are consistent with the view that chronic eosinophil mucosal inflammatory disease in NP involves a self-sustaining mechanism, i.e. local release of inflammatory mediators, independent of allergen stimulation of nasal mucosa. Increased release of inflammatory mediators contributes to the development of nasal polyps, determining oedema and an increased recruitment of inflammatory cells. Besides eosinophils, mast cells also play a key role in this process.

Age-related changes in the expression of CD95 (APO1/FAS) on blood lymphocytes.

Aging is associated with alterations of the immune system, thought to be related to an increased susceptibility to infectious diseases, and possibly to cancer and autoimmunity in the elderly. In the present paper we report data obtained on freshly collected blood from 148 healthy subjects of different ages (from cord blood to 102 years old). The subjects were divided into seven age classes (cord blood, 3-11 years, 15-39 years, 41-60 years, 61-74 years, 75-84 years, 85-102 years) and their lymphocyte subsets and the expression of the apoptosis-related molecule CD95 were evaluated. In respect of lymphocyte subsets, the major differences were found in the cord-blood samples compared with the oldest old groups. In the cord-blood group, the absolute number of all the lymphocyte subsets was enhanced, but in the oldest group, an increase of CD16+ lymphocytes was observed, whereas CD19+ lymphocytes, which progressively decrease with age, continue to decrease further in the very old. The data show that the expression of CD95 increases until age 74 years, whereas in the oldest old it tends to decrease again. The trend of CD95 expression seems to be related to the change of expression of CD95 on CD4+ lymphocytes, because the CD8+/CD95+ population rose steadily throughout the entire age range. The evaluation of CD95+/CD45R0+ lymphocytes shows similar results to those observed analyzing CD95 on total lymphocytes. Furthermore, a constant increase of CD95+/CD28+ and a related decline of CD28+ lymphocytes was observed in all age groups. These data suggest that the expression of CD95 on the different subsets of lymphocytes can be considered a good marker for studies of immunosenescence, because it may be predictive of successful aging, and can partially explain the change in lymphocytes subsets in elderly.

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.

Interleukin-12 release by mitogen-stimulated mononuclear cells in the elderly

Defects involving cellular expression of activation molecules, cell mediated immune response and natural killer (NK) activity are commonly observed in the elderly. Herein, data are reported on the evaluation of IL-12 production by old subjects. IL-12 is, actually, considered the key molecule for the induction of a T helper 1 (Th1) -type and NK response. IL-12 production from old subjects peripheral blood mononuclear cells (PBMNC) was evaluated using T-independent (bacterial lipopolysaccharide, LPS) or -dependent (phytoemagglutinin, PHA; immobilized anti-CD3 monoclonal antibodies, anti-CD3) mitogens. The IL-12 production after LPS stimulation was not reduced in cultures from old subjects when compared to that from young ones. On the contrary, IL-12 production by PHA or anti-CD3 stimulated PBMNC from old subjects was decreased. Furthermore, we have demonstrated a reduced CD40 and CD40 ligand (CD40L) expression on PBMNC from old subjects. This finding fits very well with the reduced cytokine production observed in the T-dependent stimulation systems, being the CD40-CD40L interaction mandatory for an efficient IL-12 production. All together, these results seem to suggest that defects in cell expression of activation molecules can affect the IL-12 secretion and in consequence other Th1-type cytokines.

Targeting Multiple Myeloma Cells and Their Bone Marrow Microenvironment

Abstract: Although multiple myeloma (MM) is sensitive to chemotherapy and radiation therapy, long‐term disease‐free survival is rare, and MM remains incurable despite conventional and high‐dose therapies. Direct (cell‐cell contact) and soluble (via cytokines) forms of interactions between MM cells and bone marrow stroma regulate growth, survival, and homing of MM cells. These interactions also play a critical role in angiogenesis and in myeloma bone disease. In recent years, several studies have established the biologic significance of cytokines in MM pathogenesis and delineated signaling cascades mediating their effects, providing the framework for related novel therapies targeting not only the MM cell, but also the bone marrow microenvironment.

Biological basis of the HLA-B8,DR3-associated progression of acquired immune deficiency syndrome.

The factors influencing the evolution of human immunodeficiency virus (HIV) infection are not fully known, but the host genotype undoubtedly plays a role in determining the outcome of the disease by affecting the immune response to HIV. The role of the host human leukocyte antigen (HLA) genotype in the regulation of susceptibility to HIV infection and expression has been studied extensively in different major risk groups. Certain HLA alleles and haplotypes, being associated with aberrant immune responses independently from HIV infection, have been reported to facilitate the rapid progression of disorders related to HIV infection. Particularly, the association of rapid acquired immunodeficiency syndrome (AIDS) progression with genes from the HLA-B8,DR3 haplotype has been reported by different research groups. It is well known that this haplotype is associated in all Caucasian populations with a wide variety of diseases with autoimmune features and in healthy subjects with a number of immune system dysfunctions, as a reduced production of T helper (Th)1 type cytokine. HIV infection may act on this genetic background triggering immunopathogenetic mechanisms leading to AIDS with a dominant Th2 profile as a common feature.

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.