Giuseppina Colonna Romano

Pathogenesis of autoimmune diseases associated with 8.1 ancestral haplotype : effect of multiple gene interactions

Genetic studies have shown that individuals with certain HLA alleles have a higher risk of specific autoimmune disease than those without these alleles. Particularly, the association in all Caucasian populations of an impressive number of autoimmune diseases with genes from the HLA-B8,DR3 haplotype that is part of the ancestral haplotype (AH) 8.1 HLA-A1, Cw7, B8, TNFAB*a2b3, TNFN*S, C2*C, Bf*s, C4A*Q0, C4B*1, DRB1*0301, DRB3*0101, DQA1*0501, DQB1*0201 has been reported by different research groups. This haplotype, the more common one in northern Europe, is also associated in healthy subjects with a number of immune system dysfunctions. It has been proposed that a small number of genes within the 8.1 AH modify immune responsiveness and hence affect multiple immunopathological diseases. In this paper, the characteristic features of this haplotype that might give rise to these diverse conditions are reviewed, focusing on the role of multiple gene interactions in disease susceptibility of 8.1 AH.

IL-10 and TNF-α polymorphisms and the recovery from HCV infection

Abstract Hepatitis C virus (HCV) infection becomes chronic in about 85% of infected individuals, whereas only 15% of infected people clear spontaneously the virus. It is conceivable that the host immunogenetic background influences the course of infection in term of recovery. Thus, in this study we have evaluated the effect of functionally relevant polymorphisms at tumor necrosis factor-α (TNFα, i.e., 2 biallelic polymorphisms at nt -863 and nt-308 of the promoter) and interleukin-10 (IL-10) loci (i.e., 1 biallelic polymorphism at nt -1082 of the promoter), on the clearance of HCV infection. To this purpose, we compared 18 Sicilian patients who had spontaneously recovered from previous HCV infection with 42 Sicilian patients with current HCV infection and 135 Sicilian healthy patients. The results demonstrate a decreased frequency of the -863CC TNF-α promoter genotype (involved in high production of this pro-inflammatory cytokine) and an increased frequency of the -1082GG IL-10 promoter genotype (involved in high production of this anti-inflammatory cytokine) in patients recovered from HCV infection. The evaluation of combined TNF-α and IL-10 genotypes revealed a significant increase of the “anti-inflammatory genotype” (low-TNF/high-IL-10 producers) in resolved HCV infection group compared with patients with persistent HCV infection. On the whole, our findings suggest that a genetically determined control of the HCV-induced inflammatory response may play a role in the resolution of HCV infection.

HLA, aging, and longevity: a critical reappraisal.

Despite a large number of studies, available data do not allow at present to reach definitive and clear conclusions on role of HLA on longevity, owing to major methodological problems, such as serological and molecular typing of different loci, insufficient sample sizes, different inclusion criteria and age cut-off, inappropriate mixing of data referred to people from 58 to over 100 years of age, inappropriate control matching, and neglected consideration of sex-related effects and the different genetic make-up of studied populations. However, within this confused scenario, some data emerge. First, two studies that do not fit the biases above discussed show that some HLA alleles are associated with longevity. However, some of these alleles may confer an increased risk to undergo a variety of diseases. Second, longevity may be associated with an increased homozygosity at HLA loci. Third, an intriguing association between longevity and the 8.1 ancestral haplotype (AH), which has been proven to be associated with a variety of immune dysfunctions and autoimmune diseases, apparently emerges. This association appears to be a sex-specific (males) longevity contributor, and it is particularly interesting, taking into account that a type 2 (early infancy) --> type 1 (adulthood) --> type 2 (aging) shift of cytokine profile occurs lifelong, and that individuals bearing this haplotype show a type 2 immune responsiveness (note that type 1 cytokines mainly enhance cellular responses, whereas type 2 cytokines predominantly enhance humoral responses). On the whole, the (sex specific) association of longevity with alleles or haplotypes of several genes related to risk factors for a variety of diseases (cardiovascular diseases, cancer), including HLA alleles and haplotypes, is not unexpected on the basis of previous studies on the genetics of longevity in centenarians. This association can be interpreted under the perspective of a well known evolutionary theory of aging (antagonistic pleiotropy). This theory predicts that the same gene (or allele or haplotype) can have different roles (positive or negative) in different periods of the life span. Thus, the 8.1 AH should exert a positive effect during the infancy and aging but not in adulthood, when, indeed it is associated to susceptibility to a variety of diseases.

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.

Immunogenetics of longevity. Is major histocompatibility complex polymorphism relevant to the control of human longevity? A review of literature data.

Literature data suggest that human longevity may be directly correlated with optimal functioning of the immune system. Therefore, it is likely that one of the genetic determinants of longevity resides in those polymorphisms for the immune system genes that regulate immune responses. Accordingly, studies performed on mice have suggested that the Major Histocompatibility Complex (MHC), known to control a variety of immune functions, is associated with the life span of the strains. In the last 25 years, a fair number of cross-sectional studies that searched for the role of HLA (the human MHC) genes on human longevity by comparing HLA antigen frequencies between groups of young and elderly persons have been published, but conflicting findings have been obtained. In fact, the same HLA antigens are increased in some studies, decreased in others and unchanged in others. On the whole, that could lead us to hypothesize that the observed age-related differences in the frequency of HLA antigens are due to bias. In our opinion, this hypothesis is real for most studies owing to major methodological problems. However, some studies that do not meet these biases have shown an association between longevity and some HLA-DR alleles or HLA-B8,DR3 haplotype, known to be involved in the antigen non-specific control of immune response. Thus, HLA studies in man may be interpreted to support suggestions derived from the studies on congenic mice on MHC effects on longevity. However, in mice the association may be by way of susceptibility to lymphomas whereas, in human beings, the effect on longevity is likely, via infectious disease susceptibility. Longevity is associated with positive or negative selection of alleles (or haplotypes) that respectively confer resistance or susceptibility to disease(s), via peptide presentation or via antigen non-specific control of the immune response.

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.

B cells and immunosenescence: A focus on IgG+IgD―CD27― (DN) B cells in aged humans

Immunosenescence contributes to the decreased ability of the elderly to control infectious diseases, which is also reflected in their generally poor response to new antigens and vaccination. It is known that the T cell branch of the immune system is impaired in the elderly mainly due to expansion of memory/effector cells that renders the immune system less able to respond to new antigens. B lymphocytes are also impaired in the elderly in terms of their response to new antigens. In this paper we review recent work on B cell immunosenescence focusing our attention on memory B cells and a subset of memory B cells (namely IgG(+)IgD(-)CD27(-)) that we have demonstrated is increased in healthy elderly.

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)‐induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4+ phenotype. In particular, the T helper 1‐type (Th1) subset, as evaluated by chemokine receptor‐5 (CCR5) expression, is involved in this process. Cell death in Th1‐type uses a CD95‐mediated mechanism. Furthermore, Th1‐type CCR5+ cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1‐type cells by apoptosis was confirmed by the decrease in interferon‐γ secretion. In conclusion, apoptosis of monocytes, CD4+ and CD4+ CCR5+ T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune‐depression in VL.

Cross-talk between V beta 8+ and gamma delta+ T lymphocytes in contact sensitivity.

We have previously reported that T lymphocytes proliferating in vitro to the hapten trinitrochlorobenzene (TNCB) exhibit a very restricted Vβ gene usage and response to TNCB is limited to T‐cell receptors (TCR) composed of Vβ8.2 in combination with Vα3.2, Vα8 and Vα10. This paper investigates the role played by T lymphocytes expressing the Vβ8.2 gene segment in the contact sensitivity (CS) reaction to TNCB in the intact mouse and in its passive transfer into naive recipient mice. Mice injected with monoclonal antibodies to Vβ8 are unable to develop CS upon immunization with TNCB and 4‐day TNCB‐immune lymph node cells from mice that had been depleted in vivo or in vitro of Vβ8+ T lymphocytes fail to transfer CS. However, when separated Vβ8+ and Vβ8− cells were used for passive transfer, it was found that Vβ8+ T lymphocytes failed to transfer CS when given alone to recipient mice and a Vβ8− population was absolutely required. Further analysis revealed that within the Vβ8− population, T lymphocytes expressing the γδ TCR were fundamental to allow transfer of the CS reaction. These γδ cells were found to be antigen non‐specific, genetically unrestricted and to rearrange the Vγ3 gene segment. This indicates that transfer of the CS reaction requires cross‐talk between Vβ8+ and γδ+ T lymphocytes, thus confirming our previous results obtained using TNCB‐specific T‐cell lines. Time–course experiments showed that Vβ8+ lymphocytes taken 4–24 days after immunization with TNCB were able to proliferate and produce interleukin‐2 (IL‐2) in response to the specific antigen in vitro. Similar time–course experiments were then undertaken using the passive transfer of the CS reaction system. The results obtained confirm that TNCB‐specific Vβ8+ T lymphocytes are present in the lymph nodes of immunized mice from day 4 to day 24, and reveal that γδ+ T lymphocytes are active for a very short period of time, i.e. days 4 and 5 after immunization. In fact, TNCB‐specific Vβ8+ cells are able to transfer CS when taken 4–24 days after immunization, providing the accompanying Vβ8− or γδ+ T lymphocytes are obtained 4 days after immunization. In contrast, injection of Vβ8+ T lymphocytes together with Vβ8− or γδ+ T lymphocytes that had been taken 2 or 6 days after immunization, failed to transfer significant CS into recipient mice. Taken together, our results confirm that cross‐talk between Vβ8+ and γδ+ T lymphocytes is necessary for full development of the CS reaction and may explain why the CS reaction in the intact mouse lasts up to 21 days after immunization while the ability of immune lymph node cells to transfer CS is limited to days 4 and 5 after immunization.

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.