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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The purpose of this study was to investigate the immunosenescence of skin-homing T cells expressing the cutaneous lymphocyte antigen (CLA). Peripheral blood lymphocytes from 72 healthy individuals (33 male and 39 female; median age 54 years; age-range: 18–94 years) were investigated. The expression of CD28, CD45RA and CD45RO, as well as intracellular interferon-γ (IFN-γ) and interleukin-4 (IL-4) formation of CLA+ ‘skin homing’ T cells, was analysed. In addition, T cells were detected immunohistologically in skin specimens from 15 young and 15 old, healthy individuals. The relative telomere length (RTL) was measured by fluorescence in situ hybridization using flow cytometry (flow FISH). The total number of CLA+ T cells was found to remain constant with increasing age. In contrast to peripheral blood T cells (CD3+ CLA), which showed significantly decreased CD28 and CD45RA expression in donors > 60 years of age, no age-related alterations of either CD28+ CLA+ T cells or CD45RA+ CLA+ T cells were observed. In the group of donors > 60 years of age, the proportion of intracellular IFN-γ-producing CD3+ CLA cells showed a significant increase, whereas the number of IFN-γ- and IL-4-producing CLA+ T cells was not affected by age. After stimulation with phytohaemagglutinin (PHA) or staphylococcal enterotoxin B (SEB), CLA+ T cells from old donors did not show a reduced response compared with CLA+ T cells from young donors. Additionally, the counts of T cells in healthy skin from young and old adults were statistically not different. Furthermore, the RTL was significantly shortened in enriched CD45RO+ CLA T cells from healthy old individuals, but not in aged CLA+ T cells. The present data suggest that CLA+ T cells might be a T-cell subpopulation which does not undergo immunosenescence. This may explain why the intensity of inflammatory skin reactions (e.g. psoriasis or eczema) seems to be independent of the patients' age.


Abbreviations
CLA

cutaneous lymphocyte antigen

PBMC

peripheral blood mononuclear cells

RTL

relative telomere length.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The immune system undergoes characteristic changes with ageing. T-cell function is depressed in elderly individuals, and deterioration of the immune system is believed to contribute to morbidity and mortality in humans.1

Age-associated alterations in the proportions of T-cell subsets have been well documented in humans. There are clearly a greater number of CD4+ CD45RO+ ‘memory-phenotype’ cells and fewer CD45RA+ ‘naïve-phenotype’ cells in peripheral blood mononuclear cells (PBMC) of elderly individuals.2,3 The accumulation of CD45RO+ memory cells results in a reduced ability to respond to new antigens and a retained ability to respond to recall antigens, as long as the memory cells remained present and functional.4

Thus far, the important costimulatory receptor, CD28, is perhaps the closest to a biomarker of ageing for human lymphocytes. Both in vivo and in vitro, the proportion of CD28+ cells decreases with age.5,6 Moreover, telomere lengths in CD28 cells are shorter than in CD28+ cells from the same donors, implying that the former has undergone more rounds of cell division than the latter.7 This type of proliferative senescence may therefore be responsible for the commonly observed accumulation of CD28 oligoclonal populations in elderly people.8

Data on age-associated alterations in cytokine secretion in humans are inconsistent. T helper 1 (Th1) cells are characterized by their ability to secrete interferon-γ (IFN-γ), interleukin (IL)-2, tumour necrosis factor-β (TNF-β), IL-12 and IL-15, whereas T helper 2 (Th2) cells are characterized by IL-4, IL-5, IL-6, IL-10 and IL-13 secretion. Infant humans exhibit impaired cellular, but strong humoral, immunity, and are in a type-2 dominant state. Soon thereafter, a type-1 state becomes dominant and persists in healthy humans until mid-to-later life, at which time a dominant type-2 cytokine profile may again emerge.9

Paradoxically, despite declining T-cell function, ageing does nor appear always to be associated with decreased immune reaction. There are increased levels of autoantibodies in the aged,10 resulting in autoimmune diseases (e.g. bullous pemphigoid); however, the severity of some T-cell-mediated inflammatory skin diseases, such as psoriasis, is not influenced by age.

The available data on immunosenescence of T cells in humans apply mainly to peripheral cells. The situation in the lymphoid organs is mostly unexplored, but could be different. Thus, a strong T-cell-mediated inflammatory skin reaction in old patients may be the result of differences between peripheral T cells and ‘skin-homing T cells’, which are characterized by expression of the cutaneous lymphocyte antigen (CLA).11 Almost all T cells in benign and malignant cell infiltrations of the skin express CLA on their surface.12 It was the aim of this study to investigate, for the first time, the effect of ageing on unstimulated and stimulated CLA+ T cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Study population

A group of 72 healthy individuals, 41 female and 31 male (range: 18–94 years), who fulfilled the criteria of the SENIEUR protocol,13 were selected for analysis of unstimulated T cells. For stimulation experiments, PBMC were isolated from five young (mean age: 30·4 years) and six old (mean age: 69·7 years) healthy individuals. Immunohistology was performed with healthy skin from 15 individuals < 40 years of age (mean age: 31 years) and from 15 individuals >60 years of age (mean age: 73·3 years). For determination of telomere length, 12 healthy blood donors < 40 years of age (mean age: 26·5 years) and 10 healthy individuals > 60 years of age (mean age: 72·7 years) were studied. The study was approved by the authors' Institutional Review Board.

Antibodies and reagents

All fluorescence-labelled antibodies (CD3, CD28, CD45RA, CD45RO), for fluorescence-activated cell sorter (FACS) analyses of surface molecules, were purchased from BD Biosciences (Heidelberg, Germany) or Pharmingen (Heidelberg, Germany). The antibodies for controls were fluorescein isothiocyanate (FITC)-conjugated rat immunoglobulin (Ig)M (Pharmingen) and FITC- or phycoerythrin (PE)-conjugated mouse IgG1 and IgG2, respectively. PE-conjugated mouse anti-IFN-γ and anti-IL-4 monoclonal antibodies (mAbs) were from BD Biosciences. For immunohistology, T cells were stained with anti-CD45RO mAb from DAKO (Hamburg, Germany). Staphylococcus aureus enterotoxin B (SEB) and phytohaemagglutinin (PHA), for stimulation of PBMC, were purchased from Sigma (Munich, Germany).

Isolation of PBMC

PBMC were isolated by Lymphoprep™ (Nycomed, Oslo, Norway) density-gradient centrifugation, as previously described.14 Briefly, heparinized venous blood was layered over Ficoll–sodium metrizoate and centrifuged at 700 g for 30 min. Cells at the interface above the Ficoll–sodium metrizoate were removed and washed three times with RPMI-1640.

Isolation of CLA+ and CD45RO+ CLA T cells from peripheral blood

CLA+ and CD45RO+ CLA T cells were isolated from PBMC by using an immunomagnetic separation system. T cells were separated from non-T cells with the MACS Pan T Cell Isolation kit (Miltenyi, Bergisch Glodbach, Germany). For depletion of B cells, monocytes, natural killer (NK) cells, dendritic cells, early erythroid cells, platelets and basophils from PBMC, these cells were incubated with hapten-conjugated CD11b, CD16, CD19, CD36 and CD56 antibodies and MACS MicroBeads coupled to an anti-hapten mAb. The magnetically labelled cells were depleted by retaining them on a MACS LS Separation Column in the magnetic field of the MidiMACS. The isolated T cells were resuspended in phosphate-buffered saline (PBS) and counted. Subsequently, CLA+ T cells were separated from CLA T cells by labelling the CLA+ T cells with FITC-conjugated CLA antibody and MACS anti-FITC Micro Beads. The magnetically labelled CLA+ T cells were obtained by retaining them on a MACS MS Separation Column in the magnetic field. The separation procedure was repeated once in order to achieve a higher degree of purity of CLA+ T cells, which was > 96%, as measured by FACS analysis. The remaining unbound cells were designated as CLA T cells. Purity of this T-cell population was > 98%. For measurement of telomere length, CD45RO+ CLA T cells were further enriched using a FITC-labelled CD45RO antibody (purity > 95%). The cells thus obtained were counted, resuspended in 10% dimethylsulphoxide (DMSO) and 90% fetal calf serum (FCS), and stored in liquid nitrogen.

Culture and stimulation conditions

The basic culture medium for PBMC was RPMI-1640 supplemented with 2 mm glutamine, 100 µg/ml streptomycin and 100 IU/ml penicillin. Medium containing 10% FCS is referred to as RPMI-1640 + 10% FCS.

PBMC suspensions containing 2 × 106/ml viable cells in RPMI-1640 + 10% FCS were dispensed in a volume of 1 ml to 15-ml conical polypropylene tubes. All cultures were incubated for a total of 48 hr at 37° in a humidified atmosphere of 5% CO2 in air.

PBMC (2 × 106 cells/ml) were stimulated with SEB (final concentration 5 µg/ml) and PHA (final concentration 1 µg/ml). For detection of intracellular cytokines, 20 µl of Brefeldin A (BFA; Sigma) per 1 ml of PBMC (10 µg/ml of cell suspension, final concentration) was added for the last 5 hr of activation.

Flow cytometric analysis

After purification, 5 × 106 cells were sequentially stained with FITC-conjugated anti-CLA mAb or anti-CD3 mAb, together with anti-CD28-PE, anti-CD45RA-PE or anti-CD45RO-PE. Stained cells were fixed in 2% paraformaldehyde. The controls were FITC-conjugated rat IgM and FITC- or PE-conjugated mouse IgG1 or IgG2.

For intracellular staining of cytokines in unstimulated or stimulated CLA+ and CLA T cells, 100 µl of 20 mm EDTA per 1 ml of PBMCs was added. After incubation for 15 min at room temperature, 10 ml of cold PBS was added and mixed vigorously. After centrifugation, the pellet was resuspended in 3 ml of FACS Lysing solution (BD Biosciences). Then, the cells were permeabilized for 10 min at room temperature. After this step, CLA- or CD3-FITC conjugated and PE-labelled anti-IL-4 or anti IFN-γ mAbs, as well as the appropriate isotype controls, were added and incubated for 30 min in the dark at room temperature. After washing, the cells were fixed with 200 µl of 1% paraformaldehyde solution. Flow cytometric analysis was performed using a FACScan (BD Biosciences)

Skin specimens and immunohistology

Tissue samples of normal skin were obtained from plastic surgical operations after obtaining informed consent from the patients. Sections cut at 4 µm were placed on glass slides which were then dried, deparaffinized by xylene, and rehydrated through graduated solutions of ethanol. The sections were incubated with anti-CD45RO mAb (1 : 50 dilution) for 45 min after washing with Tris-buffered-saline (TBS). The sections were then stained with the Fuchsin Plus Substrate-System™ (DAKO), following the instructions of the manufacturer. Counterstaining of the sections was performed with haemalaun. The sections were examined by conventional light microscopy (×40) and positive cells of four different fields were counted.

Telomere fluorescence in situ hybridization and flow cytometry (Flow FISH)

Telomere length of the CLA+ and CD45RO+ CLA T cells was measured by using the DAKO Telomere PNA Kit/FITC for Flow Cytometry (DAKO). Relative telomere length (RTL) was determined by comparing isolated test cells with a control cell line [1301; subline of the Epstein–Barr virus (EBV) genome negative T-cell leukaemia line CCRF-CEM].15

Test cells (CLA+ and CD45RO+ CLA T cells) and the control cells (cell line 1301) were washed in PBS and mixed 1 : 1. A total of 5 × 105 cells was resuspended in 300 µl of hybridization solution containing 70% formamide with either no probe (unstained control) or with a fluorescein-conjugated telomere PNA probe. The cells were heated for 10 min at 82° for DNA denaturation. Hybridization was performed overnight at room temperature in the dark.

Cells were washed with DAKO Wash Solution and heated for 10 min at 40°. Subsequently, cells were resuspended in 0·5 ml of DAKO DNA staining solution (a buffer containing propidium iodide and RNAse A for DNA staining) and incubated at 2–8° for 2–3 hr in the dark. Samples were then analysed by flow cytometry (FACScan; BD Biosciences) using logarithmic scale FL1-H for probe fluorescence and linear scale FL3-H for DNA staining. Samples that hybridized with the Telomere PNA Probe/FITC exhibited a fluorescence signal in FL1, which was higher than the background/autofluorescence signal obtained from the sample of the same cells hybridized with the hybridization solution without probe. Statistical data on these cells were then used to calculate the RTL of the sample cells compared with the control cells, according to the manufacturer's instructions.

Statistical analysis

Data are expressed as the mean value ± standard error of the mean (SEM). Statistical analysis for paired comparisons was conducted by using the Student's t-test. Linear regression was applied to analyse the relationship between markers of immunosenescence (CD28, CD45RA, CD45RO, IFN-γ, IL-4) and age for T cells expressing or not expressing CLA.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

CLA+ and CLA CD3+ T cells

The proportion of unstimulated CD3+ CLA T cells showed a significant decrease (P < 0·0001) in young compared with old adults, whereas the number of CD3+ CLA+ T cells was unaffected (Fig. 1a). Additionally, the number of freshly isolated and unstimulated CD28+ CLA T cells was significantly lower in old individuals, whereas the amount of CD28+ skin homing T cells remained essentially unchanged (Fig. 1b). The analysis of CD45RA+ CLA and CD45RO+ CLA T cells showed a significant decrease of ‘naïve’ T cells (CD45RA) and a significant increase of ‘memory’ T cells (CD45RO) in the population >60 years of age. In contrast, no differences between young and old in the expression of CD45RA and CD45RO on CLA+ T cells were observed (Fig. 1c, 1d).

image

Figure 1. Percentage of CD3+ T cells (a) and of T cells expressing CD28 (b), CD45RA (c), and CD45RO (d). Scatter plots show the results for all 72 healthy patients between 18 and 94 years of age. Note that linear regression analysis revealed no significant effect of age on any of the cutaneous lymphocyte antigen (CLA)-positive T-cell subsets.

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IFN-γ+ and IL-4+ CLA+ and CLA T cells

The ratio of CLA IFN-γ+/CLA IL-4+ T cells was found to increase significantly (P = 0·04) as a function of age. On the other hand, the ratio of CLA+ IFN-γ+/CLA+ IL-4+ T cells was not altered in old individuals (Fig. 2).

image

Figure 2. Ratio of freshly isolated interferon-γ (IFN-γ)+/interleukin-4 (IL-4)+ T cells expressing or not expressing the cutaneous lymphocyte antigen (CLA). Note that linear-regression analysis revealed a significant increase of the ratio for CLA T cells, but not for CLA+ T cells.

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Stimulation with PHA and SEB

After stimulation with either SEB or PHA, the number of CD3+ CLA T cells was significantly reduced in old compared with young individuals (SEB, P = 0·026, PHA, P = 0·009). In contrast, neither unstimulated (<40 years, 4·45 ± 1·84% versus >60 years, 4·36 ± 2·18%) nor stimulated (SEB, 9·43 ± 2·08% versus 9·34 ± 3·28%; PHA, 8·46 ± 2·22% versus 8·21 ± 2·96%) CD3+ CLA+ T cells were reduced in number in old individuals (Fig. 3).

image

Figure 3. Flow cytometry analysis of cutaneous lymphocyte antigen (CLA)-positive and -negative T cells (CD3+) stimulated with either phytohaemagglutinin (PHA) or staphylococcal enterotoxin B (SEB). (a) Histograms of a typical experiment with T cells from a young (28 years of age) blood donor (unbroken line) and from an healthy 93-year-old-individual (dotted line). (b) Stimulated CD3+ CLA+ and CD3+ CLA T cells from young and old healthy individuals. **P < 0·01 and *P < 0·05 compared with unstimulated cells.

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Stimulation of PBMC with SEB or PHA resulted in a significantly lower proliferation of CD28+ CLA T cells in old individuals (>60 years of age) (Table 1). However, stimulation of CD28+ CLA+ T cells revealed no significant differences between young and old probands.

Table 1.  Expression of CD28, CD45RA and CD45RO as well as the ratio of interferon-γ (IFN-γ)+/interleukin-4 (IL-4)+ T cells (CLA+ and CLA) after stimulation with phytohaemagglutinin (PHA) and staphylococcal enterotoxin B (SEB)
 CLA+CLA
<40 years>60 years<40 years>60 years
  • CTA, cutaneous lymphocyte antigen.

  • *

    P < 0·04 compared with CLA T cells from young individuals.

  • P < 0·01 compared with CLA T cells from young individuals.

CD28+
 SEB5·77 ± 1·603·73 ± 0·5467·26 ± 4·9544·72 ± 14·98*
 PHA6·65 ± 1·725·41 ± 2·5056·72 ± 6·1536·92 ± 14·60*
CD45RA+
 SEB5·85 ± 1·646·55 ± 4·5253·39 ± 6·0334·99 ± 6·59
 PHA5·25 ± 2·355·36 ± 3·2346·13 ± 4·1731·99 ± 9·94
CD45RO
 SEB6·61 ± 0·766·85 ± 3·3838·12 ± 6·1040·48 ± 8·32
 PHA4·16 ± 1·763·45 ± 2·1034·00 ± 8·2043·15 ± 9·91
IFN-γ+/IL-4+
 SEB2·28 ± 1·152·89 ± 1·926·38 ± 3·625·47 ± 2·25
 PHA2·61 ± 1·614·38 ± 2·832·69 ± 0·937·72 ± 3·95*

After stimulation with SEB, the number of CD45RA+ CLA T cells was significantly lower in the elderly compared with young adults (Table 1). No statistically significant differences were measured between stimulated CD45RA+ CLA+ or CD45RO+ CLA+ T cells from young and old individuals.

Stimulation with PHA resulted in a significant increase of the IFN-γ/IL-4 ratio in CLA T cells from old individuals (>60 years of age) compared with younger adults (<40 years of age). Again, no effects on intracytoplasmatic cytokines were observed in CLA+ T cells in adults of the older age-group (Table 1).

Immunohistology of T cells in young and old skin

In healthy, non-sun-exposed skin from individuals <40 years of age, 6·1 ± 5·98 T cells were detected. In the skin from individuals > 60 years of age, a mean of 7·1 ± 6·14 T cells was counted (Fig. 4). The differences between young and old individuals were not significant.

image

Figure 4. Numbers of T cells in healthy skin not exposed to ultraviolet (UV) light. (a) Skin from the back of a young male adult and (b) from the back of an 83-year-old man. Arrows indicate positively stained T cells in the dermis. (c) Box plots show the results of immunohistology for all individuals <40 (n = 15) and >60 (n = 15) years of age. Note that there is a moderate increase in the number of T cells counted in the skin specimens from older donors.

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Telomere length

After immunomagnetic separation, the purity of CD45RO+ CLA+ and CD45RO CLA cells was 89·8 ± 4·1% and 92·1 ± 7·1%, respectively. The RTL of enriched CD45RO+ CLA T cells from old individuals (n = 10) was significantly reduced (P = 0·02) compared with CD45RO+ CLA T cells from individuals of the younger age-group (Fig. 5). The RTL of CLA+ T cells isolated from young individuals (n = 12) was shorter than the RTL of CD45RO+ CLA T cells from the same age-group. However, the difference was not significant. No difference in RTL was detected between CLA+ T cells from young and aged healthy individuals.

image

Figure 5. Results of fluorescence in situ hybridization using flow cytometry (flow FISH) of purified CD45RO+ cutaneous lymphocyte antigen (CLA)-negative and -positive T cells from healthy individuals. (a) Isolated CD45RO+ CLA and CLA+ T cells from a young and an old individual were analyzed after hybridization with or without fluorescein isothiocyanate (FITC)-labelled peptide nucleic acid (shaded and open histograms, respectively). (b) The bar chart shows the mean ± standard error of the mean (SEM) relative telomere length (RTL) in cells of young and old donors. Note that the RTL of CLA+ T cells is lower than the RTL of CD45RO+ CLA T cells. A moderate increase of the RTL in aged CLA+ T cells is evident.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study we demonstrate that immunosenescence of CLA+ skin-homing T cells is different from that of CLA T cells. Established biomarkers of immunosenescence (such as CD28, CD45RO, CD45RA and Th1/Th2 cytokine production) were unaffected in aged CLA+ T cells, as was telomere length, indicating that CLA+ T cells may represent a non-ageing T-cell subset.

Thus far, CD28 is perhaps the closest to a biomarker of ageing found for human lymphocytes. Both in vivo and in vitro, the proportion of CD28+ cells decreases with age.6 This has been supported by our data in CD3+ CLA CD28+ cells, but not for the CLA+ population. CD28 is an important costimulatory receptor, which has important functional consequences.5 It has been suggested that the ability of centenarians' T cells to respond by medium-term proliferation to alloactivation and mitogen activation correlates with the percentage of CD28+ cells in their PBMC.6 This is confirmed by the findings of Adibzadeh et al.,16 demonstrating that the age-associated decrease in density of expression of CD28 on CD4+ T-cell clones in culture correlated with their proliferative capacity. Studies on the molecular control of CD28 expression and on the reason for an age-associated reduction in CD28 expression, suggested a loss of binding activities to at least two regulatory motifs of the CD28 promoter in T cells from the aged.17 In contrast, CLA+ T cells from aged donors showed no decreased response to either mitogen (PHA) or superantigen (SEB) induced stimulation. This indicates that CD28+ CLA+ T cells not only remain stable during life but also retain their functional capacity. Interestingly, the delayed hypersensitivity reaction to 2,4-dinitrochlorobenzene (DNCB) has been demonstrated to be impaired in previously unsensitized elderly individuals.18 Delayed-type hypersensitivity requires antigen processing by Langerhans' cells, which then migrate to the local lymph nodes where they activate T helper cells. However, in the same study it was demonstrated that reactivity to tuberculin does not show a significant fall-off with an increase in age. This might be explained by the distinction between the capacity to react to an antigen to which one has previously been sensitized (effected by ‘memory-phenotype’ T cells) and the ability to develop reactivity upon primary exposure to a new antigen. Besides the overall decrease of CLA CD3+ and CD28+ T cells in the elderly, the proportion of CD45RA+ ‘naïve’ T cells is reduced and an accumulation of CD4+ CD45RO+ ‘memory-phenotype’ cells is well documented.2,3 The latter changes are also observed in strictly selected elderly populations and seem to occur independently of health and nutritional status.19 This would be coupled with a predicted reduced ability to respond to new antigens. However, age had no impact on the numbers of either ‘memory-phenotype’ or ‘naïve-phenotype’ CLA+ T cells in our population. If the functions of aged CLA+ T cells are not altered, one may suggest that impaired sensitization of T cells in the elderly might be the result of decreased accessory cell function (e.g. cytokine production)20,21 defects in antigen processing, or in the presentation/transportation of antigens by dendritic cells (DC) to the germinal centres of lymph nodes.22

In CLA T cells, compromised function might be sought at the level of signal transduction through either or all of the T-cell receptor (TCR) components.23,24 Stimulation with the superantigen, SEB, which activates T cells via cross-linking of the MHC II and the TCR, resulted in a significantly decreased proliferation of aged CD3+ CLA T cells, but not of aged CLA+ T cells. This indicates that stimulation of CLA+ T cells from healthy elderly individuals via the TCR pathway is still intact. Additionally, mitogen-induced stimulation of CD3+ T cells, which was significantly reduced in old CLA T cells, was not reduced in CLA+ T cells from the same donors.

Moreover, the ratio of intracellular IFN-γ and IL-4 cytokine-producing CLA+ cells was not altered, whereas CD3+ CLA cells from old donors showed a significantly increased IFN-γ/IL-4 ratio compared with T cells from young individuals. Either increased25 or unchanged production of IFN-γ,26 as well as enhanced IL-4 production,27,28 has been reported in old individuals. As we have determined the proportion of T cells that are positive intracellularly for either IFN-γ or IL-4 in freshly isolated cells without in vitro stimulation, our results reflect more closely the endogenous Th1/Th2 status. Because of the conflicting results, the question of whether a type 1 or type 2 dominant state occurs in the elderly has yet to be definitively answered.

Taken together, these data clearly demonstrate that immunosenescence of unstimulated and stimulated CLA+ T cells is completely different from the CLA T-cell population. Therefore, in order to confirm the suggestion that CLA+ T cells might be a non-ageing T-cell population, telomere length was determined in CD45RO+ CLA and CLA+ T cells from young and old individuals.

Loss of telomeric DNA, and gradual shortening of telomeres, has been proposed to result, after a certain number of cell divisions, in the inability of cells to divide again.29 Loss of telomeric repeats is not an in vitro phenomenon, as it is also observed in human cells in vivo.30 Telomere length decreases as a function of donor age and thus contributes to the replicative senescence of normal cells.29,31 Telomere shortening with age was observed in PBMC populations32 and in naïve and memory CD4+ and CD8+ peripheral T cells.33 Rufer et al.34 have recently shown that total lymphocytes, as well as subpopulations of CD4+ and CD8+ T cells, showed a biphasic decline in telomere length that was rapid during early childhood and more gradual, although still highly significant, thereafter. This is confirmed by our results, demonstrating that the RTL of purified CD45RO+ CLA T cells from old donors was significantly reduced. In contrast, no shortening of RTL was observed in the enriched CLA+ T-cell population from donors up to 93 years of age. As it has been shown that telomere length in human blood cells is related to age,32 this result confirms our hypothesis that CLA+ T cells are a non-ageing T-cell subpopulation.

Immunosenescence of T cells in the lymphoid organs, including the skin, is mostly unexplored. To date it has been suggested that immunosenescence of circulating T cells, of the skin immune system and other compartments, are comparable.35 Our results clearly show that this dogma cannot be proclaimed any longer. Clinically, it is a fact that the severity of chronic T-cell-mediated inflammatory skin diseases, such as atopic eczema or psoriasis, is not altered and, for instance, adverse drug reactions are increased in old patients. This clinical observation in T-cell-mediated skin diseases can now be explained, in part, by our observation that CLA+ T cells do not appear to undergo immunosenescence. On the other hand, it has been suggested that an increased proportion of skin infections and non-melanoma skin cancer might be the consequence of an impaired skin immune system. Of course, the occurrence of cancer increases in the elderly, but a contribution of immunosenescence to this progression is difficult to ascertain. Studies that have sought to demonstrate increased cancer rates in elderly individuals with poor immune function, compared to those with good immune function, have not shown such an association, even over a 10-year follow-up period.36

These contradictions could have several explanations.

(1) Morphological and functional immunoscenesence of CLA+ T cells is different from CLA T cells and has not been recognized by the parameters we measured.

(2) Other immunocompetent cells of the skin (e.g. Langerhans' cells, keratinocytes, B cells and granulocytes) might be compromised in old individuals.

(3) Expression of E-selectin, the ligand for CLA on endothelial cells, could be compromised.

(4) Our data may reflect only endogenous ageing and ignore the influence of exogenous factors such as ultraviolet (UV) light, which undoubtedly is the most important risk factor for skin cancer.

To answer these questions, further investigations on the immunosenescence of CLA+ T cells are required.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Richard Ivell for critical reading of the manuscript. Gerlinde Finger and Birgit Mähnß are thanked for excellent technical assistance. We also thank Linda Ruhde for help with the collection of blood samples. This work was supported by grants from the Leidenberger-Müller-Foundation and from the Buch-Foundation, Germany.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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