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Early activation of γδ T lymphocytes in the elderly
Mechanisms of Ageing and Development 121 (2000) 231 – 238 www.elsevier.com/locate/mechagedev
Early activation of gd T lymphocytes in the elderly Giuseppina Colonna Romano a, Marcella Potestio a, Giuseppe Scialabba a, Andrea Mazzola b, Giuseppina Candore a, Domenico Lio a, Calogero Caruso a,* a
Dipartimento di Biopatologia e Metodologie Biomediche, Uni6ersita` di Palermo, Corso, Tukory 211, 90134 Palermo, Italy b Cooperati6a Sociale Padre Pio, Via Sal6atore Di Pasquale 8, 90011 Bagheria, Italy Received 21 July 2238; received in revised form 23 August 2000; accepted 30 August 2000
Abstract T cell function is altered in vivo and in vitro in elderly compared with young subjects, and this alteration is believed to contribute to morbidity and mortality in man due to the greater incidence of infection, as well as autoimmunity and cancer in elderly. The majority of T cells express TCRab whereas TCRgd is expressed on a minority of T cells. Moreover, it is known that gd T lymphocytes display major histocompatibility complex (MHC)- unrestricted cytotoxicity that is reminiscent of natural killer (NK) activity. In view of earlier findings on both T cells and NK cells in the elderly, we hypothesised a different behaviour of gd T lymphocytes from old subjects when compared with gd T lymphocytes obtained from young people. Therefore, to gain further insight into mechanisms of immunosenescence in this little-studied population, we studied immunofluorescence analysis gd T cells from the elderly. Our preliminary results show that the percentage of blood gd T cells in lymphocytes from old subjects is decreased when compared with the young. Interestingly, these cells are more activated in the elderly than in young subjects; expression of CD69, an early activation marker, is increased in gd T lymphocytes from old subjects after three hours of in vitro culture both with and without lipopolysaccharide stimulation. Thus, our findings, which need confirmation, strongly suggest that, in humans, gd T cells are early responders when compared with ab T cells. They may act as ‘first aid’ cells to replace the described deficit of the specific and aspecific immunity in elderly. In this view, the proinflammatory status,
* Corresponding author. Tel.: +39-09-16555911, fax: + 39-09-16555933. E-mail address: [email protected] (C. Caruso). 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 1 3 - X
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observable in the elderly, renders them ready to be stimulated by exogenous agents. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CD69; Elderly; gd cell; Immunosenescence; LPS
1. Introduction T cell function is altered in vivo and in vitro in old subjects compared with young ones and this alteration is believed to contribute to morbidity and mortality in elderly due to the greater incidence of infection, as well as autoimmunity and cancer (Pawelec et al., 1999). T lymphocytes bear T cell receptors (TCR) derived from four different rearranging genes. The TCR expressed on the majority of T lymphocytes is called TCRab whereas TCRgd is expressed on a minority of T cells (about 2–5% of peripheral T lymphocytes). The role of ab and gd T lymphocytes seems to be quite different and these differences can be also predicted on the basis of the different tissue localisation. In fact, in rodents, gd T cells are preferentially localised in epithelial tissues, and for this reason they have been proposed as a ‘first line of defence’. Moreover, cells carrying the TCRab recognise processed antigens associated with major histocompatibility complex (MHC) whereas the role of antigen processing and presentation by MHC in recognition by the TCRgd remains to be determined. An additional characteristic of gd T cells is that the majority of this subset is double negative, with a small proportion expressing CD8 and an even smaller proportion expressing CD4 (De Libero, 1997; Poccia et al., 1997; Fisch et al., 2000). It is well known that ab and gd cells cooperate (Kaufmann et al., 1993) and, by virtue of cytokines produced by gd cells, it is possible that these cells can drive ab T cell responses. In fact, it has been demonstrated that gd cells arrive earlier than ab T cells at the site of infection (Kaufmann, 1994) and in vitro studies have shown that gd T cells respond more readily than ab T cells in vitro and in vivo (Lahan et al., 1998). However, the general conclusion, in so far as one may be drawn from extensive and diverse studies, is that gd T cells may play an important role in the early stage of the inflammatory responses to many common pathogens and in the resolution of pathogen-induced inflammatory immune responses, although the nature of the ligand recognised by this TCR remains contentious. Moreover, it is known that gd T lymphocytes display MHC-unrestricted cytotoxicity that is reminiscent of natural killer (NK) activity (Poccia et al., 1997; Fisch et al., 2000). In view of earlier findings on both T cells and NK cells in elderly (Pawelec et al., 1999; Di Lorenzo et al., 1999), we hypothesised a different behaviour of gd T lymphocytes from old subjects when compared with gd T lymphocytes, obtained from young people. Therefore, to gain insight into the mechanisms of immunosenescence of these cells, we studied gd T cells from elderly by immunofluorescence analysis. First, we evaluated the percentage of gd T cells in peripheral blood of healthy young and old donors, thereafter, we focused on the ability of gd T cells to respond to lipopolysaccharides (LPS) obtained from gram-negative bacteria. Our
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results show that the percentage of gd T lymphocytes in peripheral blood of old subjects is decreased compared with young subjects. Interestingly, these cells are more activated in the elderly than in young subjects. In fact the early activation marker CD69 (Candore et al., 1995) expression is increased in gd T lymphocytes from old subjects after three hours of in vitro culture both with and without LPS stimulus. Our data suggest that gd T lymphocytes from aged individuals are in an activated condition so they are prompt in defence response. 2. Materials and methods
2.1. Subjects and analysis of lymphocytes Fifty-eight healthy old subjects (age range 70–90, 10 males, 48 females) and 38 young healthy subjects (age range 23–45, 10 males, 28 females) were studied. In each experiment all the subjects were sex-matched. In vitro activation was performed on samples from 19 old (age range 70–90) and 10 young (age range 24–32) subjects. None of the selected subjects was affected by neoplastic, infectious or autoimmune diseases, nor were any of them receiving any drug that might influence immune function at the time of the study. Informed consent was obtained according to Italian laws.
2.2. In 6itro cultures Peripheral blood collected in heparinised tubes was cultured for three hours with LPS (1 mg from E. Coli 026:B6, Sigma L-3755, Sigma-Aldrich, Milan) or with RPMI medium only, as described by Lahan et al. (1998). The samples were incubated at 37°C in an atmosphere of 95% air, 5% carbon dioxide, at 90% relative humidity. At the end of the culture time, blood was processed for flow-cytometry.
2.3. Flow-cytometry 100 ml, of freshly collected or cultured blood were incubated with the appropriate antibodies. In all the samples, lymphocyte populations were evaluated by threecolour fluorescence using the following monoclonal antibodies: anti-CD3/CD8/ CD45 (Fluorescein Isothiocyanate, (FITC)/Phycoerythin (PE)/Tri-Color(TC)), anti-CD3/CD4/CD45 (FITC/PE/TC), anti-CD3/CD19/CD45 (FITC/PE/TC) antiCD3/CD16 +56(FITC/PE) (Caltag, Burlingame, CA). ab and gd T lymphocytes were identified by anti-CD3-PE (Caltag), and FITC anti-human ab TCR antibody (Pharmingen, Los Angeles, CA) or gd TCR pan reactive antibody FITC-conjugated (clone 11F2) (Becton Dickinson, San Jose, CA). The expression of the activation marker CD69 (anti-CD69TC, Caltag) was evaluated as mean fluorescence intensity (MFI) to assess shifts in CD69 expression that depends on the amount of CD69 product expressed on the cellular membrane. In these analyses, anti-CD3-PE antibody was omitted and the percentage of gd- and gd/CD3-reactive cells was the same.
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The lymphocyte population was identified by forward and side angle scatter (FSC, SSC) on a FACScan flow cytometer (Becton Dickinson, Mountain View, Ca, USA). A red cell lysant (Ortho-mune, Ortho Diagnostic System, Raritan, NJ, USA) was used to eliminate red cells. All measurements were made with the same instrument setting and at least 104 cells were analysed using Lysis II software (Becton Dickinson). CD69/gd were analysed on 500 gd-positive events.
2.4. Statistics Since the distribution of many lymphocyte subsets is asymmetric (Caruso et al., 1997), data are given as median together with percentile P25 and 75 values and have been compared between the different groups by a non parametric test. Median is the value so positioned in the series, that there are an equal number of observations of greater magnitude and lesser magnitude. Percentile is a value in the range of a set of data, which separates the range into two groups so that a given percentage of the measures lies below this value. The values for the different groups were compared by using the Mann-Witney-Wilcoxon test. As no significant differences were observed in each group between males and females, the results were analysed from pooled data.
3. Results
3.1. E6aluation of gd T lymphocytes In all the subjects studied, the lymphocytes subsets were evaluated. The result of this analysis (data not shown) is analogous to that earlier reported (Potestio et al., 1998). The percentage of gd T lymphocytes in peripheral blood of healthy young and old subject was evaluated as described in materials and methods. The results of our analysis are shown in Table 1 where a significant (P= 0.0005) decrease of percentage value of blood gd from elderly can be observed.
Table 1 Percentage of gd T lymphocytes in freshly collected blood of young (n =38) and old (n =58) healthy subjectsa
Young Old
P25
Median
P75
2.6 1.4
3.3 2.1
6.0 3.4
a The percentage of gd T lymphocytes in peripheral blood of young and old subject was evaluated as described in materials and methods. Values were significantly different (P= 0.0005).
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3.2. Expression of the acti6ation marker CD69 In the elderly, the early activation phase of lymphocytes is impaired and a decreased expression of CD69 has been reported (Lio et al., 1996; Potestio et al., 1998; Pawelec et al., 1999). Therefore, we have focused on the early activation of gd T lymphocytes (Fig. 1 a, b). The analysis of CD69 separately performed on ab and gd T cells demonstrates that gd T cells of old subjects are able to give a higher early response when compared with gd cells from young subjects. In fact, analysis performed on 19 old and 10 young subjects show that the expression of CD69 (assessed as MFI) is higher on gd T cells of old subjects both with and without LPS stimulus. Moreover, although ab T cells are not sensitive enough to be well stimulated in a three hour culture, the MFI of CD69 in ab cells from old subjects is lower than the expression of CD69 on ab T cells from young ones. In our opinion, this observation suggests that gd T lymphocytes from old healthy individuals are in an activated condition, ready to respond to exogenous stimuli.
4. Discussion It is known that the elderly may demonstrate a reduced ability of the immune system to give an adequate response with an outcome of increased incidence of infectious diseases, cancer and autoimmunity (Pawelec et al., 1999). It has been proposed that the changes observed in aged individuals might be considered as the result of a global reshaping of the immune system, where the immune system continuously looks for possible stable points for optimal functioning (Franceschi and Ottaviani, 1997). To gain insight into the mechanisms of immunosenescence, we planned to study some aspects of gd T cells from elderly. In fact, the study of gd T cell function may be informative on the ability of aged individuals to compensate for decrease ab T cell activities in the course of microbial infection. First, we evaluated the percentage of gd T cells in peripheral blood of healthy young and old donors, thereafter, we focused on the ability of gd T cells to respond to LPS obtained from gram-negative bacteria by evaluating CD69 expression. CD69 is a cell surface protein induced during the early phase of lymphocyte activation. It is restricted to leukocytes and acts as a signal-transmitting receptor. It is quite undetectable on the cell surface of quiescent leukocytes but it is expressed at high levels in the inflammatory infiltrates of several human diseases (Santis et al., 1995) and after in vitro activation with mitogens (Candore et al., 1995; Lio et al., 1996). The highest level of CD69 expression on the cell surface is reached 24 h after activation, but mRNA peaks after three hours. Comparative analysis of activation of ab and gd T lymphocytes has demonstrated that CD69 expression is a very early event on membranes of gd T cells (Lahan et al., 1998). Our experiments show a significant decrease in the percentage of blood gd T cells in the elderly. However, as demonstrated by Lahan et al., 1998 gd T lymphocytes respond more readily to LPS than do ab T cells. Moreover, our results show that gd T cells from old subjects are more responsive than gd T cells obtained from
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Fig. 1. Expression of CD69 on peripheral blood lymphocytes from young (n =10) and old (n =19) subjects. Analysis was performed separately on ab and gd T lymphocytes. Values are given as MFI of CD69. Individual values and median are shown. Panel A depicts the MFI values of CD69 on cells cultured for 3 h in RPMI (ab old versus young: P= n.s; gd old versus young: P =0.03); Panel B depicts the MFI values of CD69 on cells cultured for 3 h in LPS (ab old versus young: P = n.s; gd old versus young: P=0.04 significant).
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young subjects. As is well accepted, deterioration of the immune system with ageing, ‘immunosenescence’, is believed to contribute to morbidity and mortality in man due to the greater incidence of infection, as well as possibly autoimmune phenomena and cancer in the aged. Dysregulation of T cell function is thought to play a critical part in these processes (Pawelec et al., 1999). In this scenario, we believe that our preliminary report on gd T cells suggests that, in aged healthy subjects, the reduced ability of other parts of the immune system may be partly balanced by a prompt involvement of gd T lymphocytes. In fact, in the elderly, these cells, although decreased in numbers, seem to be in an activated state, as shown by the expression of CD69. However, our findings, which require confirmation in more extensive studies, strongly suggest that in humans, gd T cells are early responders as compared with ab T cells. They act as ‘first aid’ cells to replace the described deficit of specific and aspecific immunity in elderly. In this view, the proinflammatory status, observable in the elderly (Franceschi and Ottaviani, 1997; Franceschi et al., 2000), renders them ready to be stimulated by exogenous agents. This finding, if supported by further data, might give a relevant role to gd T lymphocytes in the regulation of immune response in the elderly. In fact, it is known that these cells are able to produce cytokines that, in turn, regulate the immune response (De Libero, 1997; Dieli et al., 1998). Our preliminary findings are in agreement with papers that report increases of other hallmarks of ‘inflammation’, such as acute phase proteins, lipoprotein, fibrinogen and proinflammatory cytokines in the elderly, that seem to be a feature of senescence (Cohen et al., 1997; Franceschi and Ottaviani, 1997; Baggio et al., 1998; Bruunsgaard et al., 1999; Franceschi et al., 2000). Acknowledgements This work was supported by grants from MURST, Rome (60%) to G.C.R. and (40 and 60%) to C.C. We are grateful to Cooperativa Sociale Padre Pio and its scientific consulting, Raffaele D’Anna, for the collaboration. References Baggio, G., Donazzan, S., Monti, D., Mari, D., Martini, S., Gabelli, C., Dalla Vestra, M., Previato, L., Guido, M., Pigozzo, S., Cortella, I., Crepaldi, G., Franceschi, C., 1998. Lipoprotein(a) and lipoprotein profile in healthy centenarians: a reappraisal of vascular risk factors. FASEB J. 12, 433–437. Bruunsgaard, H., Andersen-Ranberg, K., Jeune, B., Pendersen, AX, Skinhoj, P., Pedersen, B.K., 1999. A high plasma concentration of TNF-a is associated with dementia in centenarians. J. Gerontol. A. Biol. Sci. Med. Sci. 54, M357–M364. Candore, G., Cigna, D., Todaro, M., De Maria, R., Stassi, G., Giordano, C., Caruso, C., 1995. T cell activation in HLA-B8,DR3 positive individuals: early antigen expression defect in vitro. Hum. Immunol. 42, 289–294. Caruso, C., Bongiardina, G., Candore, G., Cigna, D., Colonna Romano, G., Colucci, A.T., Di Lorenzo, G., Gervasi, F., Manno, M., Potestio, M., Tantillo, G., 1997. HLA-B8,DR3 haplotype affects lymphocyte blood levels. Immunol. Invest. 26, 333 – 340.
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Cohen, H.J., Pieper, C.F., Harris, T., Rao, K.M.K., Currie, M.S., 1997. The association of plasma IL-6 levels with functional disability in community-dwelling elderly. J. Gerontol. Ser. A-Biol. Sci. Med. 52, M201–208. De Libero, G., 1997. Sentinel function of broadly reactive human gd T cells. Immunol. Today 18, 22 – 26. Dieli, F., Ptak, W., Sireci, G., Colonna Romano, G., Potestio, M., Salerno, A., Asherson, G.L., 1998. Cross-talk between Vbeta 8( + ) and gamma delta +T lymphocytes in contact sensitivity. Immunology 93, 469–477. Di Lorenzo, G., Balistreri, C.R., Candore, G., Cigna, D., Colombo, A., Colonna Romano, G., Colucci, A.T., Gervasi, F., Listi, F., Potestio, M., Caruso, C., 1999. Granulocyte and Natural killer activity in the elderly. Mech. Ageing Dev. 108, 25 – 38. Fisch, P., Moris, A., Rammensee, H.G., Handgretinger, R., 2000. Inhibitory MHC class I receptors on gd T cells in tumor immunity and autoimmunity. Immunol. Today 21, 187 – 191. Franceschi, C., Ottaviani, E., 1997. Stress, inflammation and natural immunity in the aging process: a new theory. Ageing (Milano) 4, 30–31. Franceschi, C., Bonafle, M., Valensin, S., Olivieri, F., De Luca, M., Ottaviani, E., De Benedictis, G., 2000. Inflamm-aging: an evolutionary perspective on immuno senescence. Ann. NY Acad. Sci. 908, 244–254. Kaufinann, S.H.E., 1994. Bacterial and protozoal infections in genetically disrupted mice. Curr. Opin. Immunol. 6, 518–525. Kaufinann, S.H.E., Blum, C., Yamamoto, S., 1993. Crosstalk between ab T cells and gd T cells in vivo: activation of ab T cells responses after gd T cell modulation with the monoclonal antibody GL3 1993. Proc. Natl. Acad. Sci. USA 90, 9620 – 9624. Lahan, M., Kalataradi, H., Mittelstadt, P., Pflum, E., Vollmer, M., Cady, C., Mukasa, A., Vella, A.T., Ikle, D., Harbeck, R., O’Brien, R., Bom, W., 1998. Early preferential stimulation of gd T cells by TNF-a. J. Immunol. 160, 5221–5230. Lio, D., Candore, G., Cigna, D., D’Anna, C., Di Lorenzo, G., Giordano, C., Lucania, G., Mansueto, P., Melluso, M., Modica, M.A., Caruso, C., 1996. In vitro T cell activation in elderly individuals: failure in CD69 and CD71 expression. Mech. Ageing Dev. 89, 51 – 58. Pawelec, G., Effros, R.B., Caruso, C., Remarque, E., Barnett, Y., Solana, R., 1999. T cells and aging. Front. Biosci. 4, D216–269. Poccia, F., Cipriani, B., Vendetti, S., Colizzi, V., Poquet, Y., Battistini, L., Lopez-Botet, M., Fournie´, J.T., Gougeon, M.L., 1997. CD94/NKG2 Inhibitory Receptor Complex modulates both anti-viral and anti-tumoral responses of polyclonal phosphoantigen-reactive VgVd2 T lymphocytes. J. Immunol. 159, 6009–6017. Potestio, M., Caruso, C., Gervasi, F., Scialabba, G., D’Anna, C., Di Lorenzo, G., Balistreri, C.R., Candore, G., Colonna Romano, G., 1998. Apoptosis and ageing. Mech. Ageing Dev. 102, 221 – 237. Santis, A.G., Lopez-Cabrera, M., Sanchez-Madrid, F., Proudfoot, N., 1995. Expression of the early lymphocyte activation antigen CD69, a C-type lectin, is regulated by mRNA degradation associated with AU-rich sequence motifs. Eur. J. Immunol. 25, 2142 – 2146.
.
Early activation of gd T lymphocytes in the elderly Giuseppina Colonna Romano a, Marcella Potestio a, Giuseppe Scialabba a, Andrea Mazzola b, Giuseppina Candore a, Domenico Lio a, Calogero Caruso a,* a
Dipartimento di Biopatologia e Metodologie Biomediche, Uni6ersita` di Palermo, Corso, Tukory 211, 90134 Palermo, Italy b Cooperati6a Sociale Padre Pio, Via Sal6atore Di Pasquale 8, 90011 Bagheria, Italy Received 21 July 2238; received in revised form 23 August 2000; accepted 30 August 2000
Abstract T cell function is altered in vivo and in vitro in elderly compared with young subjects, and this alteration is believed to contribute to morbidity and mortality in man due to the greater incidence of infection, as well as autoimmunity and cancer in elderly. The majority of T cells express TCRab whereas TCRgd is expressed on a minority of T cells. Moreover, it is known that gd T lymphocytes display major histocompatibility complex (MHC)- unrestricted cytotoxicity that is reminiscent of natural killer (NK) activity. In view of earlier findings on both T cells and NK cells in the elderly, we hypothesised a different behaviour of gd T lymphocytes from old subjects when compared with gd T lymphocytes obtained from young people. Therefore, to gain further insight into mechanisms of immunosenescence in this little-studied population, we studied immunofluorescence analysis gd T cells from the elderly. Our preliminary results show that the percentage of blood gd T cells in lymphocytes from old subjects is decreased when compared with the young. Interestingly, these cells are more activated in the elderly than in young subjects; expression of CD69, an early activation marker, is increased in gd T lymphocytes from old subjects after three hours of in vitro culture both with and without lipopolysaccharide stimulation. Thus, our findings, which need confirmation, strongly suggest that, in humans, gd T cells are early responders when compared with ab T cells. They may act as ‘first aid’ cells to replace the described deficit of the specific and aspecific immunity in elderly. In this view, the proinflammatory status,
* Corresponding author. Tel.: +39-09-16555911, fax: + 39-09-16555933. E-mail address: [email protected] (C. Caruso). 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 1 3 - X
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observable in the elderly, renders them ready to be stimulated by exogenous agents. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CD69; Elderly; gd cell; Immunosenescence; LPS
1. Introduction T cell function is altered in vivo and in vitro in old subjects compared with young ones and this alteration is believed to contribute to morbidity and mortality in elderly due to the greater incidence of infection, as well as autoimmunity and cancer (Pawelec et al., 1999). T lymphocytes bear T cell receptors (TCR) derived from four different rearranging genes. The TCR expressed on the majority of T lymphocytes is called TCRab whereas TCRgd is expressed on a minority of T cells (about 2–5% of peripheral T lymphocytes). The role of ab and gd T lymphocytes seems to be quite different and these differences can be also predicted on the basis of the different tissue localisation. In fact, in rodents, gd T cells are preferentially localised in epithelial tissues, and for this reason they have been proposed as a ‘first line of defence’. Moreover, cells carrying the TCRab recognise processed antigens associated with major histocompatibility complex (MHC) whereas the role of antigen processing and presentation by MHC in recognition by the TCRgd remains to be determined. An additional characteristic of gd T cells is that the majority of this subset is double negative, with a small proportion expressing CD8 and an even smaller proportion expressing CD4 (De Libero, 1997; Poccia et al., 1997; Fisch et al., 2000). It is well known that ab and gd cells cooperate (Kaufmann et al., 1993) and, by virtue of cytokines produced by gd cells, it is possible that these cells can drive ab T cell responses. In fact, it has been demonstrated that gd cells arrive earlier than ab T cells at the site of infection (Kaufmann, 1994) and in vitro studies have shown that gd T cells respond more readily than ab T cells in vitro and in vivo (Lahan et al., 1998). However, the general conclusion, in so far as one may be drawn from extensive and diverse studies, is that gd T cells may play an important role in the early stage of the inflammatory responses to many common pathogens and in the resolution of pathogen-induced inflammatory immune responses, although the nature of the ligand recognised by this TCR remains contentious. Moreover, it is known that gd T lymphocytes display MHC-unrestricted cytotoxicity that is reminiscent of natural killer (NK) activity (Poccia et al., 1997; Fisch et al., 2000). In view of earlier findings on both T cells and NK cells in elderly (Pawelec et al., 1999; Di Lorenzo et al., 1999), we hypothesised a different behaviour of gd T lymphocytes from old subjects when compared with gd T lymphocytes, obtained from young people. Therefore, to gain insight into the mechanisms of immunosenescence of these cells, we studied gd T cells from elderly by immunofluorescence analysis. First, we evaluated the percentage of gd T cells in peripheral blood of healthy young and old donors, thereafter, we focused on the ability of gd T cells to respond to lipopolysaccharides (LPS) obtained from gram-negative bacteria. Our
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233
results show that the percentage of gd T lymphocytes in peripheral blood of old subjects is decreased compared with young subjects. Interestingly, these cells are more activated in the elderly than in young subjects. In fact the early activation marker CD69 (Candore et al., 1995) expression is increased in gd T lymphocytes from old subjects after three hours of in vitro culture both with and without LPS stimulus. Our data suggest that gd T lymphocytes from aged individuals are in an activated condition so they are prompt in defence response. 2. Materials and methods
2.1. Subjects and analysis of lymphocytes Fifty-eight healthy old subjects (age range 70–90, 10 males, 48 females) and 38 young healthy subjects (age range 23–45, 10 males, 28 females) were studied. In each experiment all the subjects were sex-matched. In vitro activation was performed on samples from 19 old (age range 70–90) and 10 young (age range 24–32) subjects. None of the selected subjects was affected by neoplastic, infectious or autoimmune diseases, nor were any of them receiving any drug that might influence immune function at the time of the study. Informed consent was obtained according to Italian laws.
2.2. In 6itro cultures Peripheral blood collected in heparinised tubes was cultured for three hours with LPS (1 mg from E. Coli 026:B6, Sigma L-3755, Sigma-Aldrich, Milan) or with RPMI medium only, as described by Lahan et al. (1998). The samples were incubated at 37°C in an atmosphere of 95% air, 5% carbon dioxide, at 90% relative humidity. At the end of the culture time, blood was processed for flow-cytometry.
2.3. Flow-cytometry 100 ml, of freshly collected or cultured blood were incubated with the appropriate antibodies. In all the samples, lymphocyte populations were evaluated by threecolour fluorescence using the following monoclonal antibodies: anti-CD3/CD8/ CD45 (Fluorescein Isothiocyanate, (FITC)/Phycoerythin (PE)/Tri-Color(TC)), anti-CD3/CD4/CD45 (FITC/PE/TC), anti-CD3/CD19/CD45 (FITC/PE/TC) antiCD3/CD16 +56(FITC/PE) (Caltag, Burlingame, CA). ab and gd T lymphocytes were identified by anti-CD3-PE (Caltag), and FITC anti-human ab TCR antibody (Pharmingen, Los Angeles, CA) or gd TCR pan reactive antibody FITC-conjugated (clone 11F2) (Becton Dickinson, San Jose, CA). The expression of the activation marker CD69 (anti-CD69TC, Caltag) was evaluated as mean fluorescence intensity (MFI) to assess shifts in CD69 expression that depends on the amount of CD69 product expressed on the cellular membrane. In these analyses, anti-CD3-PE antibody was omitted and the percentage of gd- and gd/CD3-reactive cells was the same.
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The lymphocyte population was identified by forward and side angle scatter (FSC, SSC) on a FACScan flow cytometer (Becton Dickinson, Mountain View, Ca, USA). A red cell lysant (Ortho-mune, Ortho Diagnostic System, Raritan, NJ, USA) was used to eliminate red cells. All measurements were made with the same instrument setting and at least 104 cells were analysed using Lysis II software (Becton Dickinson). CD69/gd were analysed on 500 gd-positive events.
2.4. Statistics Since the distribution of many lymphocyte subsets is asymmetric (Caruso et al., 1997), data are given as median together with percentile P25 and 75 values and have been compared between the different groups by a non parametric test. Median is the value so positioned in the series, that there are an equal number of observations of greater magnitude and lesser magnitude. Percentile is a value in the range of a set of data, which separates the range into two groups so that a given percentage of the measures lies below this value. The values for the different groups were compared by using the Mann-Witney-Wilcoxon test. As no significant differences were observed in each group between males and females, the results were analysed from pooled data.
3. Results
3.1. E6aluation of gd T lymphocytes In all the subjects studied, the lymphocytes subsets were evaluated. The result of this analysis (data not shown) is analogous to that earlier reported (Potestio et al., 1998). The percentage of gd T lymphocytes in peripheral blood of healthy young and old subject was evaluated as described in materials and methods. The results of our analysis are shown in Table 1 where a significant (P= 0.0005) decrease of percentage value of blood gd from elderly can be observed.
Table 1 Percentage of gd T lymphocytes in freshly collected blood of young (n =38) and old (n =58) healthy subjectsa
Young Old
P25
Median
P75
2.6 1.4
3.3 2.1
6.0 3.4
a The percentage of gd T lymphocytes in peripheral blood of young and old subject was evaluated as described in materials and methods. Values were significantly different (P= 0.0005).
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3.2. Expression of the acti6ation marker CD69 In the elderly, the early activation phase of lymphocytes is impaired and a decreased expression of CD69 has been reported (Lio et al., 1996; Potestio et al., 1998; Pawelec et al., 1999). Therefore, we have focused on the early activation of gd T lymphocytes (Fig. 1 a, b). The analysis of CD69 separately performed on ab and gd T cells demonstrates that gd T cells of old subjects are able to give a higher early response when compared with gd cells from young subjects. In fact, analysis performed on 19 old and 10 young subjects show that the expression of CD69 (assessed as MFI) is higher on gd T cells of old subjects both with and without LPS stimulus. Moreover, although ab T cells are not sensitive enough to be well stimulated in a three hour culture, the MFI of CD69 in ab cells from old subjects is lower than the expression of CD69 on ab T cells from young ones. In our opinion, this observation suggests that gd T lymphocytes from old healthy individuals are in an activated condition, ready to respond to exogenous stimuli.
4. Discussion It is known that the elderly may demonstrate a reduced ability of the immune system to give an adequate response with an outcome of increased incidence of infectious diseases, cancer and autoimmunity (Pawelec et al., 1999). It has been proposed that the changes observed in aged individuals might be considered as the result of a global reshaping of the immune system, where the immune system continuously looks for possible stable points for optimal functioning (Franceschi and Ottaviani, 1997). To gain insight into the mechanisms of immunosenescence, we planned to study some aspects of gd T cells from elderly. In fact, the study of gd T cell function may be informative on the ability of aged individuals to compensate for decrease ab T cell activities in the course of microbial infection. First, we evaluated the percentage of gd T cells in peripheral blood of healthy young and old donors, thereafter, we focused on the ability of gd T cells to respond to LPS obtained from gram-negative bacteria by evaluating CD69 expression. CD69 is a cell surface protein induced during the early phase of lymphocyte activation. It is restricted to leukocytes and acts as a signal-transmitting receptor. It is quite undetectable on the cell surface of quiescent leukocytes but it is expressed at high levels in the inflammatory infiltrates of several human diseases (Santis et al., 1995) and after in vitro activation with mitogens (Candore et al., 1995; Lio et al., 1996). The highest level of CD69 expression on the cell surface is reached 24 h after activation, but mRNA peaks after three hours. Comparative analysis of activation of ab and gd T lymphocytes has demonstrated that CD69 expression is a very early event on membranes of gd T cells (Lahan et al., 1998). Our experiments show a significant decrease in the percentage of blood gd T cells in the elderly. However, as demonstrated by Lahan et al., 1998 gd T lymphocytes respond more readily to LPS than do ab T cells. Moreover, our results show that gd T cells from old subjects are more responsive than gd T cells obtained from
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Fig. 1. Expression of CD69 on peripheral blood lymphocytes from young (n =10) and old (n =19) subjects. Analysis was performed separately on ab and gd T lymphocytes. Values are given as MFI of CD69. Individual values and median are shown. Panel A depicts the MFI values of CD69 on cells cultured for 3 h in RPMI (ab old versus young: P= n.s; gd old versus young: P =0.03); Panel B depicts the MFI values of CD69 on cells cultured for 3 h in LPS (ab old versus young: P = n.s; gd old versus young: P=0.04 significant).
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young subjects. As is well accepted, deterioration of the immune system with ageing, ‘immunosenescence’, is believed to contribute to morbidity and mortality in man due to the greater incidence of infection, as well as possibly autoimmune phenomena and cancer in the aged. Dysregulation of T cell function is thought to play a critical part in these processes (Pawelec et al., 1999). In this scenario, we believe that our preliminary report on gd T cells suggests that, in aged healthy subjects, the reduced ability of other parts of the immune system may be partly balanced by a prompt involvement of gd T lymphocytes. In fact, in the elderly, these cells, although decreased in numbers, seem to be in an activated state, as shown by the expression of CD69. However, our findings, which require confirmation in more extensive studies, strongly suggest that in humans, gd T cells are early responders as compared with ab T cells. They act as ‘first aid’ cells to replace the described deficit of specific and aspecific immunity in elderly. In this view, the proinflammatory status, observable in the elderly (Franceschi and Ottaviani, 1997; Franceschi et al., 2000), renders them ready to be stimulated by exogenous agents. This finding, if supported by further data, might give a relevant role to gd T lymphocytes in the regulation of immune response in the elderly. In fact, it is known that these cells are able to produce cytokines that, in turn, regulate the immune response (De Libero, 1997; Dieli et al., 1998). Our preliminary findings are in agreement with papers that report increases of other hallmarks of ‘inflammation’, such as acute phase proteins, lipoprotein, fibrinogen and proinflammatory cytokines in the elderly, that seem to be a feature of senescence (Cohen et al., 1997; Franceschi and Ottaviani, 1997; Baggio et al., 1998; Bruunsgaard et al., 1999; Franceschi et al., 2000). Acknowledgements This work was supported by grants from MURST, Rome (60%) to G.C.R. and (40 and 60%) to C.C. We are grateful to Cooperativa Sociale Padre Pio and its scientific consulting, Raffaele D’Anna, for the collaboration. References Baggio, G., Donazzan, S., Monti, D., Mari, D., Martini, S., Gabelli, C., Dalla Vestra, M., Previato, L., Guido, M., Pigozzo, S., Cortella, I., Crepaldi, G., Franceschi, C., 1998. Lipoprotein(a) and lipoprotein profile in healthy centenarians: a reappraisal of vascular risk factors. FASEB J. 12, 433–437. Bruunsgaard, H., Andersen-Ranberg, K., Jeune, B., Pendersen, AX, Skinhoj, P., Pedersen, B.K., 1999. A high plasma concentration of TNF-a is associated with dementia in centenarians. J. Gerontol. A. Biol. Sci. Med. Sci. 54, M357–M364. Candore, G., Cigna, D., Todaro, M., De Maria, R., Stassi, G., Giordano, C., Caruso, C., 1995. T cell activation in HLA-B8,DR3 positive individuals: early antigen expression defect in vitro. Hum. Immunol. 42, 289–294. Caruso, C., Bongiardina, G., Candore, G., Cigna, D., Colonna Romano, G., Colucci, A.T., Di Lorenzo, G., Gervasi, F., Manno, M., Potestio, M., Tantillo, G., 1997. HLA-B8,DR3 haplotype affects lymphocyte blood levels. Immunol. Invest. 26, 333 – 340.
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