spontaneous or natural killer cytotoxicity of k562 ... · james e. nagel, gary d. collins, and...

[CANCER RESEARCH 41, 2284-2288, June 1981]0008-5472/81 /0041-0000$02.00

Spontaneous or Natural Killer Cytotoxicity of K562 ErythroleukemicCells in Normal Patients

James E. Nagel, Gary D. Collins, and William H. Adler1

Clinical Immunology Section, Gerontology Research Center, National Institute on Aging. NIH, Baltimore, Maryland 21224

ABSTRACT

Peripheral blood mononuclear cells from 200 normal individuals, ages 20 to 95 years, were evaluated for their capabilityto mediate spontaneous or natural killer (NK) cytotoxicity usingthe K562 erythroleukemic cells as targets. The results of a 4-hr 51Crspecific release assay demonstrated that the NK activity

of normal individuals is independent of age, sex, and smokinghabits. Although varying greatly among individuals, the NKactivity of 25 persons restudied after a mean 20-month interval

remained stable. Deficient NK lytic activity is not characteristicof elderly individuals.

INTRODUCTION

The in vitro lysis of tumor cells by lymphocytic effector cellsmay proceed through the activities of at least 3 types ofcytotoxic mononuclear cells: cytotoxic T-lymphocytes; antibody-dependent killer cells; and antibody-independent NK.2

Among this functional classification, NK putatively have theunique ability to recognize, without prior sensitization, nonan-tibody-coated targets and to spontaneously lyse these allo-

genic, syngeneic, and xenogeneic tumor cells. This capabilityto detect and eliminate nonspecifically foreign cells on initialexposure has led to the proposal that NK function as theprimary effectors of immunosurveillance (15, 24, 33). Althoughknown to be nonphagocytic, nonadherent, and heterogeneousfor the presence of Fc receptors for IgG, further morphologicalcharacteristics as well as the origin of human NK have notbeen fully defined. Increasing experimental evidence indicatesthat both the spontaneous (NK) and antibody-dependent cel

lular cytotoxic activities of peripheral blood cells are mediatedby the same subpopulations, which have been characterizedby various investigators to be a T-lymphocyte, promonocyte,or "null" cell or to be of myeloid origin (21, 22, 32, 39).

Many T-cell-mediated immune functions diminish with ad

vancing age; however, the effect of aging on NK activity isunclear. For example, in mice and rats in which the age-related

change is most easily demonstrated, the NK activity of spleencells is absent in the very young, peaks at 5 to 8 weeks of age,and then decreases thereafter. Most studies of human NKactivity have indicated that reactivity does not diminish withage; however, many published investigations have dealt withsmall numbers of individuals or have studied NK activity in ill ortumor-bearing patients of various ages (8, 20, 30, 37, 46, 47).

Despite the lack of conclusive evidence demonstrating an in

1To whom requests for reprints should be addressed.2 The abbreviations used are: NK, natural killer cell(s); BLSA, Baltimore

Longitudinal Study of Aging; RPMI, Roswell Park Memorial Institute; PCS, fetalcalf serum.

Received October 20, 1980; accepted March 2, 1981.

vivo cause and effect relationship between diminished NKactivity and neoplasia, there are sufficient data from the experimental mouse model to support the conclusion that, undercertain circumstances, NK are important in resistance to tumorgrowth (1, 13, 33). The proposed immunosurveillance role ofNK, coupled with recent reports on vital statistics demonstrating that death from malignant neoplasms ranks second tocardiovascular disease as the leading cause of death in individuals over 65 years of age, prompted the present investigation of a large group of well-characterized normal adults on the

effect of age on human NK activity.

MATERIALS AND METHODS

Study Population. The study population was composed of200 adults, ages 20 to 95 years, who are participants in theBLSA. The study group that is reported here included 50females and 150 males. The BLSA is an investigation of normative human aging which began for males in 1958. A companion program for females was initiated in 1978. The self-recruited participants voluntarily return approximately every 18to 24 months to undergo a 3-day inpatient rÃ©Ã©valuationof

psychological, biomÃ©dical, and physical changes. Essentially,all participants are ambulatory, and most live in their ownresidence with a spouse. Stone and Norris (43) have summarized previously the demographic and social characteristics ofthe male participants. The male subjects included in this reporthave been reevaluated an average of 8 (range, 2 to 19) timesand the female participants have been reevaluated twice. TheBLSA program does not make any overt attempts to alter thelife style or living habits of the participants, other than to advisethe subjects and their personal physicians of any significantmedical abnormalities which may be detected during theirperiodic evaluation.

For the purposes of this study, smokers were defined asthose individuals who have a history of smoking at least 10cigarettes/day. Nonsmokers were considered to be those whowere not smoking at the time of their evaluation, but this groupdoes include former smokers.

Isolation of Mononuclear Cells. Heparinized peripheralblood (20 ml) was diluted 1:4 with RPMI Medium 1640 (GrandIsland Biological Co., Grand Island, N. Y.) and separated bydifferential buoyancy centrifugation on a Ficoll:sodium diatri-

zoate cushion (LSM; Litton Bionetics, Kensington, Md.). Theinterface cells were removed with a Pasteur pipet and washedtwice with RPMI Medium 1640, and the mononuclear cell countwas determined in a hemocytometer.

Cytototoxicity Assay. Cultured cells from the K562 erythroleukemic cell line (provided by Dr. R. B. Herberman, NationalCancer Institute, NIH) grown in RPMI Medium 1640 supplemented with 10% PCS, 10 rriM, 4-(2-hydroxyethyl)-1-piperazi-

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NK Activity and Aging

neethanesulfonic acid, 2 mM L-glutamine, and 100 /ig genta-

mycin per ml were used as target cells. The line was routinelysubcultured the day before use in an assay. Prior to an assay,the target cells (2 x 106 in 0.15 ml of Tris buffer, pH 7.4) wereincubated with occasional mixing with 0.2 /iCi of Na251CrO4 in

0.2 ml of sterile isotonic 0.9% NaCI solution (Amersham/SearleCorp., Arlington Heights, III.; specific activity 50 to 100 Ci/mgchromium) for 30 min at 37Â°. The chromium-labeled K562

target cells were then centrifuged twice through 2 ml of heat-inactivated PCS (to remove unincorporated 51Cr), then resus-

pended in RPMI Medium 1640 containing 10% PCS, andcounted. After adjusting the density to 1 x 10s cells/ml in

RPMI Medium 1640 with 25 mM 4-(2-hydroxyethyl)-1 -pipera-zineethanesulfonic acid and 10% heat-inactivated PCS, 100/il (10" radio-labeled target cells) were dispensed into each

well of a round-bottom microtiter plate (Linbro Chemical, New

Haven, Conn.) containing 100 /il of the serially diluted effectorcells. Eight target:effector ratios from 1:100 to 1:0.78 wereused. Each ratio was run in duplicate. Following the addition ofthe target cells, the microtiter plates were centrifuged for 3 minat 400 x g and then incubated at 37Â° in a humidified atmo

sphere containing 5% COj:95% air. After 4-hr incubation, 100

/il of supernatant were removed from each well and placed inindividual 12- x 75-mm glass tubes, and the radioactivity was

determined in a gamma counter (Nuclear Chicago, Des Plaines,III). The percentage of specific 51Cr release was calculated as

% of releaseE-S

W- S x 100

where Â£is cpm in the experimental tube, S is cpm spontaneousrelease, and W is cpm maximal isotope release effected bywater lysis. Dose-response curves were obtained for eachindividual by plotting the percentage of specific 51Cr release

obtained at each of 8 effectortarget ratios and the logic of thenumber of effector cells. The mean spontaneous 51Cr release

by the radiolabeled K562 targets in the absence of effectorcells was 7.1%.

RESULTS

Effect of Age and Gender on NK Activity. The NK activityof peripheral blood lymphocytes from 200 individuals (50 females and 150 males) against K562 target cell is shown inChart 1. Data analysis by least-squares linear regression of thepercentage of 51Cr release for the entire group demonstrated

no significant differences (at the p < 0.05 level) in NK activityas a result of advancing age. Since the NK activity of eachindividual was tested at 8 effectortarget ratios, a comparisonwas made of the slopes of the linear portions of the semilog-arithmic plot of effector cell number and percentage of 51Cr

release. A linear regression model was used to evaluate thesignificance of each regression coefficient with those (5 of200) not meeting the p < 0.05 level of significance beingexcluded. Analysis of covariance by age group demonstratedequality of the regression lines across age groups, indicatingno significant age-related change in peripheral blood NK activ

ity.Chart 2 displays a comparison of the peripheral blood NK

activity of males and females according to the age of the celldonors. There were no significant differences (t test) in periph-

20-2930-394M9 50-59SM9 70-7980-95

n- 15 41 24 35 39 32 14

AGEIYEARSI

Chart 1. Geometric mean percentage of 51Cr release at a 5:1 effectontarget

ratio as a function of cell donor age. Radiolabeled K562 target cells wereincubated 4 hr as described in "Materials and Methods." Number of individuals

in each age group is represented by n.

20-2930-394049 50-5960-697079 80-95

AGEIYEARSI

Chart 2. Geometric mean percentage of 5'Cr release at a 5:1 effectortarget

ratio by age group and sex. By f-test analysis between the group means, therewas no difference by sex within each age group.

eral blood NK activity of males or females in this investigation.Because data collection was accomplished over a period of

approximately 2 years, we evaluated our results for the presence of uncontrolled variables which may have arisen duringthe study. This was done by considering the first and last 100individuals tested as separate groups and subjecting the datato an analysis of variance test. The results showed that thepopulation means of each group for both age and NK activityhad F values of <3.5 and therefore were not statisticallysignificantly different.

Smoking Status of Cell Donors of Peripheral Blood NKActivity. Previous reports indicated that NK activity may bealtered by cigarette smoking (7). Of the 200 individuals studied,41 were found to be active cigarette smokers at the time theywere tested. There were no significant differences found in themean peripheral blood NK activity of smokers and nonsmokers.However, the smoking group had by f-test analysis a slightlylower mean age (49.3 years) than did the nonsmokers (55.6years, p < 0.05), a factor which might obscure differences inperipheral blood NK activity based on smoking habits.

Longitudinal Follow-up of NK Activity. Chart 3 displays theresults of 2 measurements of peripheral blood NK activity onthe same individuals separated by a 13- to 25- (mean, 19.3)month interval. Although the group was small (n = 25) andthere was considerable variation among individuals in the levelsof their NK activity, no statistical difference by paired f-test

JUNE 1981 2285



J. E. Nagel et al.

M

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^ Â»

Ãˆ 20

10

10 20 30 Â« 50

TEST2PfRCENT[Â»&)RELEASE

Chart 3. Percentage of 51Cr release at a 5:1 effectortarget ratio of cells from

the same individual tested on 2 separate occasions. Mean interval between thetests was 19.3 months (range, 13 to 25 months). By paired ( test, there were nosignificant differences in NK activity in this time interval.

analysis was found between the first and second determinationof peripheral blood NK activity for these individuals.

DISCUSSION

In the present study, the effect of age on levels of humanperipheral blood cell NK activity was studied. Using a well-characterized group of 200 normal individuals 20 to 95 yearsof age, a statistically significant age- or sex-related difference

in peripheral blood NK activity against the K562 cell line couldnot be demonstrated. Although insignificant, it should be notedthat cells from individuals between 40 and 80 years of age didmanifest decreasing levels of cytotoxic activity. In contrast,cells from individuals over 80 years of age displayed greaterlevels of mean NK activity as compared with younger agegroups. It is important to recognize that this observation isbased on cross-sectional analysis of a population and that theolder individuals, especially those over 75 years of age, constitute a biologically selected population. These elderly subjects represent the survivors from a group which has alreadyexperienced greater than 75% mortality. If in vivo NK activityconveys a protective function via immune surveillance or by anassociation with other unidentified risk factors, it could beexpected that individuals with low NK activity would have beeneliminated by death from a population of elderly subjects. Thedeviation of the downward trend in cytotoxic activity may inferthat the elderly subjects represent the more "fit" survivors

from a larger group of individuals.Although a substantial amount of data exists indicating that

the NK activity of murine spleen cells declines with advancingage, considerable variation is present among different strainsof genetically inbred mice (18). Different patterns of an age-

associated decline in NK activity of mice and rats are alsoobserved when different cell types are used as targets (2, 23).For instance, in the rat when carcinoma or sarcoma cells, asopposed to lymphoid cells, are used as targets, constant levelsof NK activity are maintained through 22 months of age (2).Other animals, such as the pig, hamster, and guinea pig,although less extensively studied, have not demonstrated anage-related decline in NK activity (44). Prior studies of humanNK activity have, in general, demonstrated no age-associated

change, although some reports, not using K562 cells as targets, have noted an increase in NK activity with the age of thedonor (8, 20, 27, 30, 37, 46, 47). Human peripheral blood NKactivity has been reported to be influenced by gender (30, 37),smoking habits (7), histocompatibility haplotypes (31, 40),chronic alcohol consumption (42), and recent viral infection(16); making the singular effect of age on human NK activitydifficult to isolate in a cross-sectional study. In addition, indi

vidual levels of human NK activity, even among similarly agednormal subjects, are variable. Published information concerning serial measurements of individual cytotoxic activity, incontrast to the finding of the percentage study, has indicatedthat NK activity of cells from an individual fluctuates considerably (14, 36).

It has also been demonstrated in both animals and humansthat NK activity may be augmented by type l Interferon or typel Interferon inducers (5, 6, 19, 25, 41). Since studies in themouse have shown previously that advancing cell donor ageresults is enhanced Interferon production by mitogen-stimu-lated spleen cells, differences in the ability to synthesize inter-feron may be an important factor influencing or regulating thelevels of NK activity of different aged individuals (12). Certainly,Interferon is important in the modulation of NK cytotoxic activity; however, the finding of enhanced Interferon production inaging mice would appear to contradict the reported age-relateddecline in their spleen cell NK activity. The variability of theage-dependent decline of NK activity in mice, the peak and

persistance of NK activity in older mice (9, 10, 48), and theobservation that mice housed in different environments mayhave greatly different levels of NK activity (3, 28) suggest thatthe question of the age-related decline in murine NK activity

should perhaps undergo a reexamination.Although identification of cells mediating human peripheral

blood NK activity is incomplete, there is general agreementthat the cells are nonphagocytic and nonadherent, lack surfacemembrane immunoglobulin, and bear receptors for sheeperythrocytes and the Fc portion of IgG (reviewed in Refs. 18and 33). Because of the observation that several of thesecharacteristics are also shared by killer cells mediating antibody-dependent cellular cytotoxicity and because NK and killer

cells cannot be physically separated from each other, therehas been considerable speculation that both NK and killercytotoxic activities are mediated by a common cellular subpop-ulation. Whether these effector cells belong to a population ofprethymic T-cells, promonocytes, "null cells", or another cell

population remains controversial. Recent data using OKT andOKM hybridoma antibodies and laser flow cytometry suggestthat both NK and killer effector cells are not T-cells but are of

a myeloid origin (22) and are included in the subpopulation of"null" cells (29).

The relationship between NK activity and primary immuno-surveillance is an unproven but interesting hypothesis. Although the cytotoxic NK has many attributes which would allowit to fulfill a role as a primary defense mechanism against smallnumbers of tumor cells, in vivo evidence of such a role inhumans has not and probably cannot be conclusively demonstrated. Most data supporting such a surveillance role for NKhave been developed in mice where 3 observations wouldseem to be the most germane, (a) When tumor cells aretransplanted into young and old recipients, there is a lowertumor incidence in young mice with high NK activity than in old




NK Activity and Aging

mice with diminished NK activity (11). (Jb) There is a higherincidence of spontaneous tumors and leukemias among strainsof mice with genetically low NK activity compared with strainshaving high NK activity (48). (c) NK activity may be increasedby the injection of tumor cells (16). The possible relationship ofthese findings to the increased incidence of neoplasia in elderlyhumans without demonstrably lower levels of NK activity remains speculative. While the present cross-sectional investi

gation has demonstrated that peripheral blood NK activity didnot diminish with age or gender, it is important to consider 2elements of its experimental design which may be relevant tothe results. Since only one cell type, the K562 erythroleukemiacell line, was used as a target, it is important to note that in themouse model, spleen cell NK activity directed against targetsof either solid tumor or lymphoid origin displays different age-

related patterns. Another important consideration is that, sinceour study did not evaluate the NK activity of individuals whowere less than 20 years of age, the possibility exists that peakNK activity may occur in younger individuals, and the levelsseen in our study group represent decreases from earlier peakactivity. This possibility has been suggested by results of otherstudies (8).

Several additional points should also be emphasized. Assuggested by findings from our laboratory and others whichindicate that peripheral blood NK activity may be altered byenvironmental factors, diet, and viral infections, a measurementof the ability to generate an NK cytotoxic response to anappropriate stimulus may be more critical than quantitation ofa "basal" level of NK activity in peripheral blood. This would

appear, in particular, to be the case if the modulation of NKactivity by interferon is found to be related to specific in vivopathological conditions. Other reports indicating that the augmenting effects of interferon on NK activity occur by recruitment of precursor cells from the bone marrow (38) and thatcyclic nucleotides may modulate NK cytolysis (34) suggest avariety of other sites at which age-related changes in function

could effect NK activity. Studies that compare human NKactivities against allogeneic and autologous tumors point outthat a critical point in tumor immunosurveillance may be thecapacity of the NK effector cell to recognize the tumor cellantigenic determinants (26). Although distinct target structureshave been reported on lymphoma cell lines (35), their role inNK-mediated cytolysis has been questioned (4). Whether there

are alterations in tumor recognition ability that are secondaryto age-related changes is unknown (45). An additional investi

gation of all of these points will be needed to answer theimportant question of the relevance of NK cytolysis to thefrequent occurrence of neoplasia in the elderly.

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1981;41:2284-2288. Cancer Res James E. Nagel, Gary D. Collins and William H. Adler Erythroleukemic Cells in Normal PatientsSpontaneous or Natural Killer Cytotoxicity of K562

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