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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2410-2420
Age-Related Decline of Perforin Expression in Human Cytotoxic T
Lymphocytes and Natural Killer Cells
By
Daniel Rukavina,
Gordana Laskarin,
Gordana Rubesa,
Natasa Strbo,
Ivica Bedenicki,
Darko Manestar,
Mario Glavas,
Stephen E. Christmas, and
Eckhard R. Podack
From the Department of Physiology and Immunology, Medical Faculty,
University of Rijeka, Rijeka; the Department of Otorhinolaringology,
Clinical Hospital Center, University of Rijeka, Rijeka, Croatia; the
Clinical Hospital Center Pula, Pula, Croatia; the
Department of Immunology, University of Liverpool, Liverpool, UK; and
the Department of Microbiology and Immunology, University of Miami,
Miami, FL.
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ABSTRACT |
In this study a flow cytometric technique for detecting cytoplasmic
perforin (P) has been used to quantify age-related changes in perforin
expression in human peripheral blood lymphocytes (PBL). Proportions of
P+ lymphocytes increased after birth, but declined
rapidly after the age of 70 years. This was true for both T cells and
CD16+ and CD56+ natural killer (NK) cells.
Children showed in addition to high levels of perforin positive
CD8+ cells a much higher proportion of
CD4+P+ cells than the other age groups. In
elderly individuals there was also a highly significant reduction in
mean levels of perforin per cell as compared with all other groups
(P < .05 to .001). Adult women had consistently higher mean
levels of perforin per cell than adult men for all P+
cell phenotypes. Functional tests clearly showed the deficiency in
early spontaneous cytotoxic potential of PBL from elderly persons due
to relative P deficiency, which can be corrected by stimulation of
cytolytic cells with target cells and interleukin-2 (IL-2). The
deficiency in cytolytic activity on the contact with target cells may
have implications for antiviral and antitumor immunity in elderly
persons.
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INTRODUCTION |
CYTOTOXIC LYMPHOCYTES or killer cells are
the mediators in cell-mediated cytotoxicity (CMC) reactions. They
comprise cytotoxic T lymphocytes (CTL) and natural killer (NK) cells.
CTL are a part of the adaptive immune system and NK cells of the innate
or natural immune system. Both CTL and NK cells are endowed with a
potent cytolytic machinery located in the cytoplasmic granules
containing the cytolytic molecule perforin (perforating protein) and
associated granule proteases (granzymes).1,2 Perforin
"perforates" target cell membranes by forming transmembrane
pores, which lead to the equilibration of ionic gradients, normally
maintained by cell membrane, osmotic lysis, and nuclear
disintegration.3 The formation of membrane pores by
perforin is also a prerequisite for apoptosis of target cells, in the
case of successful membrane repair, stimulated by calcium ion influx.
Granzymes, which are also located in the secretory granules of
cytotoxic lymphocytes, gain entry into the target cell through perforin
pores and mediate target cell DNA degradation through the activation of
caspase 3, which is the protease responsible for cleavage of poly
(adenine diphosphate [ADP] ribose)
polymerase.4-7 The pathophysiologic significance of
perforin-mediated cytotoxicity was confirmed in experiments with
perforin-deficient animals, showing its role in protection against some
viral infections, intracellular parasites, tumors, and
allotransplants (for review, see Kagi8).
During postnatal life, from soon after birth until old age, the immune
system in humans passes through many changes both in the numbers of
immunocompetent cells and their phenotypic characteristics, as well as
functional capabilities. Absolute numbers of leukocytes decline
progressively, as do total lymphocytes, T, B, and NK
cells.9,10 These changes are accompanied by a marked
increase in the numbers of activated T cells (CD3+HLA
DR+), as well as cells with potential to mediate non-major
histocompatibility complex (MHC)-restricted
cytotoxicity.11 NK activity assessed by cytotoxicity
against the K-562 cell line is extremely low in the neonatal period,
but increases rapidly to almost reach adult levels between 1 and 5 months of age and remains at that level throughout
childhood.12
The age-related decline in the functional activity of CTL and NK cells
has been recognized for many years. It has been demonstrated in mice
that deterioration at the molecular level (perforin and granzymes) in
the lytic mechanism may at least in part be responsible for the decline
in CTL activity.13-16 However, there are almost no data on
perforin protein expression in human peripheral blood cytolytic cells
and their subsets during various periods of the postnatal life. The
results obtained in this investigation point to both the serious
deficit of perforin expression in old and the deficiency in spontaneous
NK cytotoxicity. The significance of this finding for the functional
reactivity of cytolytic cells is discussed.
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MATERIALS AND METHODS |
Subjects.
The study was approved by the Ethics Committee of the Medical Faculty
University of Rijeka. For flow cytometric analyses, five groups were
formed. Cord blood (CB) was obtained within an hour of normal vaginal
delivery (n = 5). Children were normal healthy persons (n = 7; four
females, three males; age = 5 years) attending regular health control
clinic, and informed consent of their parents was obtained. Adults
comprised normal laboratory donors; males were aged between 20 and 31 years (n = 9) and females were aged between 20 and 34 years (n = 12).
Elderly people were normal apparently healthy individuals living in an
old people's home, were aged between 73 and 77 years (n = 7; four
females, three males), and informed consent was obtained. Proliferation and cytotoxicity assays were performed in adults and elderly persons only. For mixed lymphocyte reaction (MLR), six adults and six elderly
persons were included and for cytotoxicity assays, five elderly persons
and six adults.
Lymphocyte preparation.
Between 3 mL (children) and 10 mL of heparinized venous peripheral
blood was layered onto Ficoll/Hypaque density gradient (Nycomed Pharma AS, Oslo, Norway) and centrifuged for 20 minutes at
800g. Cells accumulating at the interface were washed twice in
RPMI 1640 and resuspended at a final concentration of 1 × 106 peripheral blood lymphocyte (PBL)/per sample in buffer
for flow cytometry. Cell viability was checked by trypan blue.
Monoclonal antibodies (MoAbs).
A murine antihuman perforin MoAb  G9 (MoAb; IgG2b) was purified from
Balb/c ascites.17 The following MoAbs were obtained conjugated to phycoerythrin from Becton Dickinson (Mountain View, CA):
Leu-4 (anti-CD3), Leu-3a (anti-CD4), Leu-2a (anti-CD8), Leu-11b (anti-CD16), and Leu-19 (anti-CD56).
Simultaneous detection of cell surface and intracellular antigens by
flow cytometry.
Our procedure for the simultaneous detection of intracellular perforin
and cell surface membrane antigens has been described elsewhere.18 Briefly, PBL were cultured overnight at
37°C in complete RPMI 1640 medium containing 10% fetal calf serum
(FCS), 2 mmol/L L-glutamine, 100 IU/mL penicillin, 100 µg/mL
streptomycin and 5 × 10 5 mol/L
2-mercaptoethanol. Nonadherent cells were aliquoted (106
per aliquot) and washed in FACS buffer (2% FCS, 1 mmol/L EDTA, 0.1%
NaN3 in phosphate-buffered saline [PBS], pH 7.4). They
were then fixed in 100 µL PBS containing 4% paraformaldehyde, pH
7.4, for 10 minutes at room temperature. After two washes in FACS
buffer, the cells were permeabilized with saponin buffer (0.1% saponin [Sigma, Poole, Dorset]), 2% goat serum and 1 mmol/L EGTA in PBS for
20 minutes at room temperature. Antiperforin MoAb in saponin buffer (3 µg/100 µL) was added to the cell suspension at a final concentration of 8 to 10 µg per sample and incubated for 30 minutes at +4°C. Second antibody (fluorescein-conjugated goat antimouse IgG;
Becton Dickinson) was added for a further 30 minutes at +4°C, with
this and subsequent rinsing steps being performed in saponin buffer.
Cell surface antigens were then labeled by incubating for 30 minutes at
+4°C with phycoerythrin-conjugated MoAbs after the integrity of the
membranes was restored by a 10-minute incubation in FACS buffer. In
some experiments, only cell surface labeling was performed. An
irrelevant isotype-matched murine MoAb was used as a negative control.
Lymphocytes were gated on the basis of forward and side scatter (FSC
and SSC). A minimum of 104 cells were analyzed on a FACScan
(Becton Dickinson), and results are shown as contour graphs. Thresholds
for positive staining were set at less than 2% using the negative
control and percentages of positive cells were obtained by subtracting
the value of the negative control.
Cell proliferation assays.
PBL from adults and elderly people (responding cells) were separated by
Ficoll-Hypaque, as described. Allogeneic stimulating cells were buffy
coat cells obtained from blood volunteer donors from the Transfusion
Center. After the washing procedure, stimulating cells were placed in
50-mL plastic culture flasks (Grainer, Frickenhausen, Germany) in RPMI 1640 supplemented with 5% FCS (GIBCO,
Gaithersburg, MD), L-glutamine (175 µg/L), tiamulin (100 mg/L),
penicillin (500 IU/L), gentamycin (2 mg/L), and streptomycin (60 mg/L).
The cells were cultured overnight at 37°C in a humidified
atmosphere with 5% CO2 to allow adherence. The adherent
cells (perforin negative) were removed by short-term (3 minutes)
trypsin treatment. The process of trypsinization was stopped by adding
10% RPMI 1640. The population of cells obtained was
mitomycin-C-treated and served as stimulating cells in one-way MLR.
The MLR was performed by adding 1 × 106 PBL to 1 × 106 mitomycin-C-treated adherent cells and
incubated either for 24 or 72 hours, when both the percentage of
perforin positive cells and the content of perforin/cell (mean
fluorescence intensity [MFI]) were measured. PBL from
adults and elderly persons were cultured in high interleukin-2 (IL-2)
(100 U/mL) medium (human rIL-2) for 6 and 24 hours, respectively, and
perforin content was measured.
Cytotoxicity assay.
Functional NK cell assays were performed with PKH-26 (orange)-labeled
K562 target cells following the manufacturer's instructions (Sigma
Biosciences, St Louis, MO; PKH-26 Red Fluorescent Cell Linker Kit). PBL
from adults or elderly persons were incubated for 2 hours with K-562
target cells prestained with PKH-26, a lipophilic dye that binds to the
cell membrane and fluoresces orange. Briefly, 1 × 105/mL-labeled K562 cells were incubated with PBL effector
cells at different killer to target ratios in a final volume of 200 µL, for 2 hours in short-term assays and 18 hours for long-term assays at 37°C in a 5% CO2 atmosphere. After washing
in FACS medium, 200 µL of propidium iodide (concentration 10 µg/mL)
was added and the percentage of killed cells was measured by flow
cytometry. Killed cells were enumerated by detecting cells that have
both orange (PKH-26) and red (propidium iodide) fluorescence.
Immunocytochemistry.
Perforin was detected in cytospins of PBL from adults and elderly
persons. Briefly, lymphocytes, washed twice, were centrifuged onto
glass microscope slides. After drying at room temperature, the cells
were fixed in cold acetone for 10 minutes and washed once in Tris
buffered saline (TBS). All incubations were performed at
room temperature in a humidified atmosphere. Endogenous peroxidase activity was blocked by H2O2, and nonspecific
binding was stopped by preincubation with 1% bovine serum albumin
(BSA). The primary antibody (mouse antihuman perforin) was applied at a
concentration of 30 µg/mL for 1 hour. Peroxidase-labeled antimouse
IgG (Boehringer, Mannheim, Germany) was used at a
dilution of 1:100 for 45 minutes. After two washing steps in TBS, the
reaction was developed by 3-amino-9-ethyl carbazole (Sigma). Nuclei
were counterstained with hematoxylin, and slides were mounted in
gelatine-glycerol.
Statistical analysis.
Results were analyzed using the Sigma Plot for Windows, version 1.02 (Jandel Scientific, Chicago, IL). Statistical analyses were performed using a Student's t-test one-way analysis for
comparison of means.
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RESULTS |
Total perforin positive cells in CB lymphocytes and PBL.
We have investigated the percentage of isolated lymphocytes containing
the cytolytic protein perforin in their cytoplasmic granules.
Approximately 12.2% of umbilical cord lymphocytes were perforin
positive (Fig 1) suggesting that fetal PBL
are endowed with cytolytic molecules, at least by the end of fetal
life. Significantly higher percentages of PBL are perforin positive in
children, not only when compared with CB (P < .001), but with
other groups (P < .05), and particularly to the aged group
(P < .001). Levels of perforin expression in PBL were
substantially lower in aged persons than in all other groups. There was
no difference between men and women.
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| Fig 1.
The percentage of total perforin positive cells (mean ± standard error [SE]) in CB lymphocytes (A,  ;) and PBL
from children (B,  ), men (C,  ), women (D,  ), and
elderly persons (E,  ). Levels of significance: (B, C,
D) versus (A, E) P < .001; (A) versus (E) P < .05;
(B) versus (C, D) P < .05.
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As explained in Materials and Methods, in all groups, cells were
double-labeled simultaneously for perforin (intracellular antigen) and
for a panel of cell surface antigens characteristic for T lymphocytes
(CD3, CD4, CD8) and NK cells (CD56, CD16).
Staining for perforin expression in double-labeled cells from various
groups as determined by two-color analysis is shown in
Fig 2. The gates were set by
using labeled isotype antibody controls for each group. Perforin
expression is represented in three different ways.
Double-positive PBL.
In this representation, double-positive cells (perforin positive and
surface marker positive) are represented as a fraction of total PBL
counted (10,000 cells), which is set at 100%
(Fig 3).
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| Fig 3.
The percentage of double positive
(perforin+ and surface marker+) lymphocytes
(mean ± SE). CB lymphocytes (A, ;) and PBL from children (B, ),
men (C, ), women (D, ), and elderly persons (E, ). Levels of
significance (from P < .05 to P < .001) for: CD3
= (A) versus (B, C, D); (B) versus (D); (B, C, D) versus (E); CD4 = (B) versus (A, C, D, E); CD16 = (B) versus (A, C, D, E); (A) versus
(E); (C) versus (D, E); (D) versus (E); CD8 = (A) versus (C, E); (B,
C, D) versus (E); CD56 = (A) versus (C, E); (C) versus (D); (B, D)
versus (E).
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In the CB lymphocytes, two thirds of perforin positive cells belong to
the NK subset (CD16; CD56; approximately 9% of cells) and one third to
T lymphocytes (CD3+; approximately 5%).
CD8+P+ are the predominant subset among
perforin positive T lymphocytes (5.3%), and
CD4+P+ cells account for only 0.8%.
Percentages of CD16+P+ and
CD56+P+ cells in this group are almost
identical, indicating that almost all CD16+ cells are also
CD56+P+.
Notable differences were found between PBL from children and all of the
other groups in that the percentages of CD3+P+
and CD16+P+ cells were significantly higher
than in any other group. Furthermore, the most prominent finding in
this group is a highly significant increase of
CD4+P+ cells (8.9% of total PBL), which is 6 to 10 times higher than in any other group (P < .001). In all other groups, CD4+P+ cells were
very low, approximately 1% of total PBL. In children, the percentage
of CD16+P+ cells (25.6%) was significantly
higher than in any other group.
In adults, the only difference between women and men was for cells of
NK phenotype. The level of CD16+P+ and
CD56+P+ cells was significantly higher in men
than in women. Furthermore, the percentage of
CD56+P+ cells in men (19.7%) was the highest
in this investigation, significantly higher than in all other groups.
The percentages of total T cells and CD8+ cells that were
perforin positive were by far the lowest in old persons, as were percentages of NK subpopulations of perforin positive cells
(CD16+P+ and CD56+P+).
Frequency of perforin expressing cells in the various lymphocyte
subsets.
The following data present perforin expression as the fraction of cells
with a certain phenotype, eg, CD3. The percentage of perforin positive
cells then represents a fraction of all CD3+ cells
(Fig 4). Both cytotoxic T lymphocytes and
NK cytolytic cells from CB lymphocytes are endowed with similar
proportions of cells with the cytolytic molecule perforin as adults.
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| Fig 4.
The frequency of perforin expressing cells (mean ± SE)
in the various lymphocyte phenotypes in CB lymphocytes (A, ;) and PBL
from children (B, ), men (C, ), women (D, ), and elderly
persons (E, ). Levels of significance (from P < .05 to
P < .001) for: CD3 = (A) versus (E); (B) versus (C, D, E);
(C, D) versus (E); CD4 = (B) versus (A, C, D, E). CD8 = (A) versus (C, E); (B) versus (E); (C, D) versus (E); CD16 = (A)
versus (B); (B) versus (C, D, E); (C, D) versus (E). CD56 = (A)
versus (B, C, E); (B) versus (D, E); (C) versus (D, E); (D) versus
(E).
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The highest level of perforin expression in T lymphocytes was found in
children (31.3%), which is significantly higher than in all other
groups. Careful analysis of this result shows that the perforin
positive cells in the CD4+ cytolytic T lymphocytes subset
(CD4+P+) contributes most of this increase. The
percentage of perforin positive cells among CD8+cells is
close to levels detected in both groups of adults, but their frequency
in CD4+cells (21.3%) is five to eight times higher than in
the other groups.
The percentage of perforin positive cells among CTL
(CD3+P+ and CD8+P+) is
the lowest in old persons. Similarly, by far the lowest levels of
perforin positive NK cells (CD16+ = 19.1%;
CD56+ = 26.3%) were found in this group (Fig 4).
There was no difference in perforin expression in CTL subpopulations
between men and women. The only difference was for the CD56+ subset with significantly higher expression in the
men's PBL (91.2% of cells perforin positive v 77.7% in
women). This is the highest proportion of perforin positive cells in
any cell population measured and indicates that in men almost all
CD56+ cells contain perforin.
Level of perforin protein expression in T and NK cell subsets.
The procedure of double-labeling of PBL for simultaneous detection of
intracellular protein (perforin) and cell surface antigens by flow
cytometry, besides enabling the detailed analyses of perforin expressing cells, also permitted the measurement of MFI, which is
supposed to be proportional to the number of perforin molecules present
in the cells. By applying standard parameters for FSC, SSC,
fluorescence intensity 1 (FL1), and FL2, we analyzed MFI for perforin in subsets of T lymphocytes
(CD3+P+, CD4+P+,
CD8+P+) and NK cells
(CD56+P+, CD16+P+)
(Table 1).
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Table 1.
The Levels of P Expression (MFI values) for CTL
(CD3+P+) and Their Subpopulations
(CD4+P+ and
CD8+P+), and NK Subpopulations
(CD16+P+ and
CD56+P+) in CB Lymphocytes and PBL From
Children, Men, Women, and Elderly Persons
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Perforin content in CB cells, for almost all subsets of T lymphocytes
and NK cells, was generally similar to values obtained in adults. PBL
of children had quantities of perforin similar to those of adults. The
content of perforin was higher in all subsets of women's PBL compared
with those from men, and the difference is significant for all subsets
of T cells and for CD16+ NK cells.
By far the lowest levels of perforin were found in all subsets of PBL
from old persons, which were significantly lower than in other groups.
Cytotoxicity assays.
In a short-term (2 hours) cytotoxicity assay, PBL from aged persons
have reduced cytotoxicity at all effector versus target ratios compared
with adults (Fig 5). There was no
difference in cytotoxicity between the 2- and 18-hour tests for adult
persons. However, prolonged effector to target contact (18 hours) of
PBL from aged persons greatly increased their cytotoxic activity, which
reached the level of adults. The deficiency of aged PBL in short-term
assay was partially corrected after 24 hours IL-2 culture, however the
cytotoxicity was still lower than in adults (Fig 5). IL-2 culture also
increased the cytotoxic potential of adults' PBL.
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| Fig 5.
NK cytotoxicity of PBL from elderly persons increased in:
(a) short-term assay, after previous IL-2 culture and (b) after
prolonged contact (18 hours) with the target K-562 cells. Results are
expressed as mean values, and each point is the average value of five
to six samples.  , short-term assay (2 hours); , short-term assay
(2 hours) with PBL cultured in IL-2 for 24 hours;  , long-term assay
(18 hours).
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The analyses of the frequency of CD8+P+ and
CD56+P+ cells in elderly persons showed
significant increases for both after 6 hours culture in IL-2 (P < .01) and approached the levels in adults. Prolonged culture in IL-2
(24 hours) further increases these values (P < .001), but
particularly MFI for perforin in both CD8+ and
CD56+ cells in old persons (Fig
6). Because the percentages of single positive CD8+ and
CD56+ cells were not changed after 6 and 24 hours IL-2
culture (not shown), it is concluded that the proportion of cells that
were double positive (CD8+P+ and
CD56+P+) increased greatly.
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| Fig 6.
Changes in the percentages and mean content of perforin
(MFI) in subpopulations of perforin positive PBL
(CD8+P+ and
CD56+P+) cultured for 6 or 24 hours in high
IL-2 medium. Results are expressed as mean values, and each point is
the average value of five to six samples. Perforin positive cells:
adults, ; elderly persons, . MFI: adults, ; elderly persons,
.
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Immunocytochemistry.
Immunocytochemistry clearly confirmed the results of FACS analyses
(Fig 7). PBL from elderly persons (Fig 7C)
exhibited very few and more lightly stained or smaller P+
intracytoplasmic granules/cell compared with adults (Fig 7A). However,
PBL of elderly persons after 24 hours IL-2 culture (Fig 7D) had
numerous P+ granules, which were stained very strongly, and
the number of granules was even higher than in the corresponding group
of adults (Fig 7B).
Proliferation assay.
Alloantigens were found to be very strong stimulators of perforin
expression in PBL of old persons. Short-term culture (24 hours)
strongly increased both the percentage of perforin positive cells and
MFI for perforin, even to higher levels than in adults (Fig 8). Both the percentage of perforin
positive cells and MFI for perforin in aged persons were significantly
higher (P < .01) after 24 hours culturing, compared with the
initial values. In adults, these values were almost unchanged. In all
groups, values for day 3 culture decreased significantly.
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| Fig 8.
Changes in the percentage of perforin positive cells and
mean content of perforin per cell in PBL from elderly persons cultured
with mitomycin-C-treated allogeneic cells. Results are expressed as
mean ± SE and each point is the average value of six samples.
Perforin positive cells: adults, ; elderly persons, . MFI:
adults, ; elderly persons, .
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DISCUSSION |
Previous reports have shown a steady decline in absolute numbers of
both CD4+ and CD8+ T-cell subsets with
advancing age,11,19 and this is reflected by a poorer
response to mitogen.19 Superimposed on this decrease is a
substantial increase in the proportion of T cells that express HLA-DR,
a marker of T-cell activation. However, this is not associated with any
change in the proportions of CD45RO+ cells,20
and there are no data to indicate whether these cells are activated
functionally. A more pronounced decrease with age in absolute B-cell
numbers was also noted,11 paradoxically associated with
increased serum levels of some Igs.21
However, absolute NK cell numbers did not decline with age and
proportions of lymphocytes expressing CD16, CD56, or CD57 were significantly increased.11 Despite these relative
increases, NK cell function on a per cell basis was either stable up to
the age of 7022 or declined with age.23 When
centenarians were analyzed as a separate group, they were found to have
higher NK and redirected killing activity than middle-aged
donors,11 but individuals surviving so long may not be
representative of the elderly population as a whole.
Recently, we have been using extensively a cell membrane
permeabilization method, adapted for flow cytometry, for investigations of perforin expression in various physiologic and pathologic
conditions, including pregnancy, allotransplantation, and
autoimmunity.18,24,25 Sumner et al,26 whose
basic procedure we followed with some modifications, were able to
directly correlate flow cytometric fluorescence intensities (MFI) with
the absolute numbers of enzyme (beta-glucuronidase) and receptor
molecules present in the T lymphocytes, thus establishing unequivocally
the quantitative validity of the flow cytometry procedure at the
molecular level. Both approaches, the detection of the percentages of
perforin positive cells and of the mean perforin content per cell, were
used in this investigation. In the latter case, MFI for perforin in
different cell populations may not be strictly proportional to the
number of perforin molecules per cell, as there are no direct data
about the penetration of antiperforin antibody into the core of the
cytoplasmic granules of permeabilized cells.
In unstimulated PBL, perforin is constitutively expressed in NK and
 / T cells (high level of expression) and in the subpopulation of
the CD8+ cells expressing the CD11b antigen (moderate
expression). However, perforin can be induced in the CD8+
CD11b subpopulation and in some CD4+ T
cells.27 Perforin expressing CTL and NK cells are not
homogenous cell populations, as we have suggested
previously.28 CTL effectors can belong to class I and class
II MHC-restricted subpopulations, ie, CD8+P+
and CD4+P+, and NK effectors to
CD56+P+ and CD16+P+
subpopulations. MHC nonrestricted natural cytotoxic killing is characteristics of lymphokine-activated killer (LAK) T
cells, CD3+TCR+CD4CD8P+
cells and a subpopulation of
CD3+  TCR+CD4
CD8, but P+ cells.29,30
Perforin expression in the CB lymphocytes and adult PB lymphocytes was
studied by Berthou et al31 and our data are largely in
agreement. Small differences could be a result of differences in the
procedures used for simultaneous staining of intracellular and cell
surface antigens. We have found that one third of perforin positive CB
lymphocytes belong to T lymphocytes (approximately 5% of all cells),
which is slightly higher than Berthou et al31 obtained
(1%). The values are almost identical when perforin positive CD3
low+ and CD3+ cells from the investigation
cited are taken together ( 6%). However, we did not perform triple
staining as they did and showed that CD3 low+ cells are a
subpopulation of NK cells. CB lymphocytes are endowed with mean levels
of perforin per cell, which are close to those seen in adults.
Therefore, our results also confirm that at least at the end of the
pregnancy, the fetal immune system is endowed with a potent cytolytic
armamentarium.
The most interesting result in children was the significantly higher
level of perforin positive cells compared with all other groups. This
is ascribed primarily to the unexpectedly high levels of
CD4+P+ cells (10% of total lymphocytes) and
additionally to the significantly higher levels of
CD16+P+ cells. As mentioned, the
CD4+P+ subset represents a subset of
CD4+ T lymphocytes with cytotoxic potential and rapid
granule-mediated killing of target cells bearing MHC class II molecules
and the appropriate antigen. It has been shown that most human
CD4+ CTL specific for human immunodeficiency virus
(HIV) are perforin positive,32 as well as
some of thyroid infiltrating lymphocytes in Hashimoto's
thyroiditis.29 Recently, we have found an increase of this
subpopulation in the acute phase of immunologically mediated kidney
allograft rejection,24 in the active phase of disease in
patients with multiple sclerosis,25 and in pathologic
pregnancies.33 Very recent results from one of our
laboratories34 offer an additional possible explanation of
the role this cell subset could play in the downregulation of the
afferent arm of the immune response through elimination of
antigen-presenting cells. The immune system in early postnatal life is
overwhelmed with new pathogens, and CD4+P+
cells could be very useful in the downregulation of immune response through elimination of antigen-presenting cells, and in that way preventing immunologically mediated self-destruction. Alternatively, CD4+ cytotoxic T cells may be instrumental in eliminating
virus-infected class II MHC expressing target cells, as children are
exposed to many novel viruses within the first few years of life.
The CD16 molecule (IgG FcRIIIA receptor) is a conventional
transmembrane protein, with a distinct cytoplasmic
domain.35 FcRIIIA is expressed on NK cells36
and links cellular and humoral immunity by serving as a bridge between
specific antibody specificity and cytotoxic effector cell function.
FcRIIIA is exclusively responsible for antibody-dependent
cell-mediated cytotoxicity (ADCC) and the CD16 NK
subset lacks this function.37 ADCC plays a key role in
immune defense against infectious diseases and in immune surveillance against malignant growth (for review, see Deo et al38). A
role of lymphocyte-mediated ADCC in the children's group is suggested by the highest level of CD16+P+ cells in this
age group, emphasizing the underlying importance of the combination of
innate and adaptive immunity at the time of definitive maturation of
the immune response. Fc receptors are of crucial importance in
directing the uptake and destruction of viruses, bacteria, and a
variety of infections and parasites and are involved in
antibody-dependent killing of infected cells expressing viral
antigens.38 FcRIIIA-expressing NK cells isolated from
HIV-seropositive individuals have been shown to be coated with anti-HIV
antibodies and readily mediate lysis of HIV-infected or gp-120-coated
target cells in vitro. Furthermore, this ADCC activity correlates
inversely with disease progression.39
There was no difference seen between men and women when total perforin
positive cells and double positive T lymphocytes and their subsets were
analyzed. However, significant differences appeared in the percentages
of double positive NK cells (both CD16+P+ and
CD56+P+ cells), which were significantly more
abundant in men (Fig 3). Conversely, in women, the levels of perforin
expression per cell (MFI) were higher in all subpopulations of double
positive T and NK cells, and the difference was significant, except for
CD56+P+ cells. Substantial decreases of
CD16+P+ cells were seen in PBL in the first
trimester of human pregnancy, followed by great increases (to a
significantly higher level than in nonpregnant women) at the end of the
pregnancy.18,33 Because CD16+P+
cells are almost absent from the pregnancy decidua (2% of decidual lymphocytes), despite the fact that 46% of decidual lymphocytes are
CD56+P+ cells, it could be concluded that
downregulation of perforin expression in the CD16+ cell
subset could have a very important physiologic role in early pregnancy,
when the fetoplacental allograft is most sensitive to the maternal
rejection reaction. We have also found differences in NK subpopulations
in multiple sclerosis (MS) patients. In the active phase
of MS, a significant increase of CD16+P+ and
decrease of CD56+P+ cells was found, which was
accompanied by a sevenfold increase of CD16+ cells among
P+ cells.25
There are very few data about the perforin expression in lymphocyte
subsets in aged humans.40 In experiments in mice, the age-related decline in CTL activity was associated with a decreased proportion of killer cells among the target-binding
population.13 The expression of perforin and granzymes
genes was decreased in primary allogeneic CTL generated from spleen
cells of aged compared with young mice. Furthermore, aging compromises
the release of granzymes by CTL in mixed lymphocyte
culture.14,15
A crucial point in the investigation of age-related decline of immune
reactivity is the question whether aging affects the functional
reactivity by decreasing the frequency of precursor cells within a
given cell population and/or by decreasing the lytic potential
of individual cells from the given cell population.
The senescent decline of CTL activity in mice was investigated by
Horvath et al16 by detecting the expression of perforin proteins at the cellular level to determine whether perforin is expressed by fewer cells or whether expression is reduced in all cells,
or both. A consistent decrease in the number of perforin positive
granules, their size, and intensity of staining was found in aged mice.
Furthermore, when stimulated in mixed lymphocyte culture, the cells
from young mice showed much higher lytic activity (approximately
sevenfold) and total P content (approximately 12-fold), while little
difference was observed in the frequency of P+ cells
(approximately twofold).16 The results of FACS analyses presented in this report clearly show that both the frequency of
perforin positive cells and the content of perforin per cell are
significantly lower in both T-cell and NK cell subsets of aged persons,
compared with all other age groups. In three independent experiments,
we have found in elderly persons significantly lower levels of both the
percentage of P+ cells and MFI for P compared with adults.
The substantial reduction in the average P content per cell (judged by
MFI) among effector cell populations in elderly persons might be
responsible for the deficient lytic potential of these cells in
short-term assays by limiting perforin-mediated lysis, which is one of
the critical steps in cytolytic action.2 In our opinion,
this deficiency in early spontaneous cytotoxicity (short-term assay) is
the main difference in the NK cytotoxic potential of PBL from old
persons compared with adults. This was unrecognized previously, as most
investigations were performed using 18-hour assays,22 and
our results clearly demonstrate the difference between 2-hour and
18-hour tests. Short-term contact is quite sufficient for adult PBL to
express their full cytotoxic potential. Moreover, our preliminary
results showed that in adults even 30 minutes is sufficient for
effector cells to exert a cytotoxic effect, although of smaller
magnitude (not shown). Prolonged contact (18 hours) of PBL from elderly
persons with K562 target cells probably helps to upregulate the
cytolytic machinery of effector cells, enabling them to reach the
cytotoxic potential of adults.
Perforin and granzyme mRNA expression in human PBL are powerfully
induced by IL-2 stimulation.41,42 There are also
experimental data showing that P appears to be the limiting factor in
NK-mediated cytolysis of NK sensitive targets in mice. Lymphocytes from
perforin-deficient mice have no cytotoxic activity against YAC targets,
while heterozygous perforin-deficient NK cells have about 50% of the
lytic activity of wild-type cells.43 Our results clearly
show that PBL from elderly persons have preserved the potential for
upregulation of P protein expression in both CTL and NK cells and
cytotoxic activity (Figs 5 and 6), after culture in 100 U/mL of IL-2.
The effector populations from the same subjects used in
the NK cytotoxicity experiments shown in Fig 5 were also
used for analyses of perforin content in cell subsets shown
in Fig 6. Accordingly, this increase in cytolytic cells and
molecules correlates well with the increase in NK cytolytic
activity of PBL from elderly persons. In the context of our study, it
appears that in elderly people P is limiting possibly due to limiting
IL-2.
It has been shown recently by Hamann et al44 that P
containing CD8+ cells are effector T cells, while neither
naive nor memory cells have significant perforin. They showed that
CD8+ T cells with low P content
(CD8+CD45RARO+) correspond
to the memory type population, which does not exert cytolytic activity
without prior in vitro stimulation, and this population could be a
candidate for the predominant CD8+ population in elderly
persons. Significant increases in the number of P+ cells
and content of P per cell following 1-day MLR culture indicate that
alloantigens are very strong stimulators of P expression in PBL of
elderly persons.
It is well known that CMC is an important mechanism of tumor control in
vivo, mediated by both CTL and NK cells, and both types of cells
predominantly use the perforin-dependent pathway.45 By
using perforin-deficient mice and an MHC class I-syngeneic lymphoma,
Van den Broek et al46 showed that NK-mediated tumor growth
control in vivo is perforin-dependent. Taking into account that
perforin is also the major effector pathway used by CTL, it is possible
that the high incidence of tumors in old age could partly be a result
of a compromised early spontaneous cytotoxicity mediated by perforin.
In this context, it would be of great interest to analyze perforin
expression in centenarians, as many immunologic parameters in this
group of exceptional individuals are much better than in other elderly
people.47
| |
FOOTNOTES |
Submitted October 9, 1997;
accepted May 26, 1998.
Supported by Grant No. 062001 from the Ministry of Science and
Technology of the Republic of Croatia and by Grants No. CA39201-15 and
CA57904-06 from the National Institutes of Health, Bethesda, MD.
Address reprint requests to Daniel Rukavina, MD,
Department of Physiology and Immunology, Medical Faculty, University of
Rijeka, B. Branchetta 20/1, HR-51000 Rijeka, Croatia;
e-mail:Daniel.Rukavina{at}mamed.medri.hr.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Ivana Godnic for secretarial help and
Davorka Percinic, Nadia Peraic, and Ksenija Tulic for technical assistance.
| |
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Abstract
Introduction
Abstract
