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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,
1071-412X/98/$04.0010
Jan. 1998, p. 105–113 Vol. 5, No. 1
Copyright © 1998, American Society for Microbiology
Immune Function in Healthy Adolescents
JACQUELINE A. BARTLETT,
1
* STEVEN J. SCHLEIFER,
1
MELISSA K. DEMETRIKOPOULOS,
1,2
BEVERLY R. DELANEY,
1
SAMUEL C. SHIFLETT,
1
AND STEVEN E. KELLER
1,2
Department of Psychiatry, UMDNJ-New Jersey Medical School,
1
and Department of Neuroscience,
UMDNJ-Graduate School of Biomedical Science, Newark,
2
New Jersey
Received 12 March 1997/Returned for modification 20 May 1997/Accepted 23 September 1997
In the present study, we examine immunological functioning in normal healthy African-American and
Latino/Latina adolescents recruited from an inner-city high school and an inner-city clinic. A battery of tests
was performed with enumerative and functional measures which encompassed both innate and adaptive
immunity. We found immune differences related to age, gender, and race on both the enumerative and the
functional immune measures. This data expands the available body of information concerning normal immu-
nity in healthy adolescents.
The immune system has most often been studied in relation
to disease, and much of the normative data has been compiled
by considering various control groups in different studies. Fur-
ther, while there is considerable data on immune parameters in
Caucasian adults, there is much less data available for other
age groups, such as adolescents, and for other ethnic groups.
Some researchers have reported normative immune data
when studying disease processes in adolescent populations (3,
11, 31, 35). However, since the focus of these studies was not
normal subjects, the description of the normal controls as well
as the small number of subjects limit the usefulness of this
work for providing normative data. More recent studies have
begun to provide some selective normative immunological data
with normal, healthy adolescent subjects. For example, in one
study enumerative (cell phenotype) data from 112 predomi-
nantly Caucasian (74%) healthy adolescents (ages 12 to 19)
(40) was examined, while in another study the functioning of
polymorphonuclear leukocytes in 58 children (aged 6 months
to 15 years) (15) was examined. Although these studies are
limited by the fact that they were focused on a single immu-
nological variable, they are a useful beginning in understanding
normal adolescent immune status.
The present study was part of a longitudinal study of behav-
ior, mood, and immunity in inner-city minority adolescents.
We evaluated 206 healthy African-American and Hispanic ad-
olescents, utilizing fresh blood cells and a battery of immune
assessments which provides data on enumerative and func-
tional immunological measures. The measures included total
leukocyte count, counts of both granulocytes and lymphocytes,
and counts of subsets of the lymphocyte populations, including
those shown to have implications in certain disease states such
as human immunodeficiency virus illness. The functional mea-
sures that were chosen involve in vitro assays only. Therefore,
no exposure to antigen or other invasive procedure was nec-
essary. This was thought to decrease subject risk and increase
participation of subjects.
This data is characterized in terms of the relationships to
age, gender, and race within this selected population of healthy
inner-city youth. Normative data from this type of under-stud-
ied population has become increasingly important with regard
to immune-related diseases. Since the advent of diseases such
as AIDS, the need for knowledge of immunity in adolescents as
well as the need for following the progression of this or other
disease states in this population has increased.
MATERIALS AND METHODS
Subjects. This study was approved by the Institutional Review Board of
UMDNJ-New Jersey Medical School. Informed consent from subjects 18 years
of age and informed assent from subjects under 18 years of age with informed
consent from a parent or guardian were obtained. A total of 331 adolescents
ranging from 12 to 18 years of age participated in the present study. All subjects
were recruited as part of a project assessing behavior, immunity, and health. Two
hundred thirteen subjects were randomly recruited from a local public high
school. One hundred eleven consecutive adolescents who were attending an
adolescent medicine clinic for a routine physical examination or follow-up for a
minor medical condition were also recruited. Seven subjects were peer referrals.
All psychosocial (e.g., age and race) and substance use (e.g., alcohol and to-
bacco) data were obtained in an interview format.
A medical history, review of systems, and vital signs were obtained. Potential
subjects were excluded if they had chronic diseases likely to have substantial
effects on immunity (e.g., neoplastic, endocrine, or immune disorders) or if they
were taking medications with known immunologic effects. Subjects with acute
infections were deferred from study until their symptoms were resolved. Subjects
presenting with other medical conditions, such as recently resolved minor infec-
tions, or with other past disorders with possible immune effects (e.g., asthma)
were studied but not included in these analyses. The occasional adolescent with
clinically apparent mental retardation, significant neurologic deficits, schizophre-
nia, or substance abuse or dependence disorders was excluded.
Medical group evaluation. Each subject was screened by a trained research
assistant with the Health Symptoms Checklist (18). Vital signs were collected at
the time of venipuncture. All medically relevant data was reviewed by the phy-
sicians (J.A.B., S.J.S., and B.R.D.) who made the final consensual determination
of the subjects’ medical status. Subjects were classified into one of the following
three medical groups: (i) healthy (subject had no medical problems and was not
taking any medication); (ii) minor medical problem (subject had mild medical
symptoms such as a cough or runny nose, had had fever within the past week
[venipuncture was deferred in subjects with current fever], or had taken medi-
cation for a cold within the past 2 weeks); or (iii) medical problem (subject was
found to have more significant chronic or current medical problems considered
likely to be associated with altered immunity). This last group consisted mainly
of asthmatics who, in the past year, had had an asthmatic attack or utilized
antiasthmatic medication and of adolescents with a history of a recent infection
requiring antibiotic therapy. Of the total 331 adolescents studied, 206 were
classified in the healthy group prior to the analysis of any data. Only the subjects
in the healthy group were included in the analyses to be described.
Immunological evaluation. All assays were carried out blind to the subjects’
medical status. Blood samples were collected in a heparinized syringe (preser-
vative-free heparin). All the results herein were obtained from the same veni-
puncture for each subject. Total leukocyte and differential counts were per-
formed by standard techniques. Phenotypic analysis of lymphocytes, monocytes,
and granulocytes was performed with heparinized whole blood. Mononuclear
cells were separated from whole blood by centrifugation on a Ficoll-Hypaque
gradient. These cells were used to assess mitogen-induced lymphocyte stimula-
tion and natural killer (NK) cell function. Additionally, granulocytic function was
* Corresponding author. Mailing address: Department of Psychia-
try, Administration Complex, Bldg. 14, UMDNJ, New Jersey Medical
School, 30 Bergen St., Newark, NJ 07107-3000. Phone: (201) 972-6385.
Fax: (973) 972-8305. E-mail: bartleja@umdnj.edu.
105
determined from granulocytes which were isolated from the remaining erythro-
cytes by a percoll gradient.
Cell phenotypes. Cell phenotypes were assessed by flow cytometry with the
Epics 1 Profile Plus (Coulter Immunology, Hialeah, Fla.). All monoclonal anti-
bodies were directly conjugated and obtained from Coulter Immunology. The
antibodies were all used in concentrations of 10 ml per 0.10 ml of whole blood.
Q-prep technology was then utilized to process the samples for cytometry.
Briefly, 50 ml of heparinized whole blood was added to 200 ml of the appropriate
antiserum or antiserum combination. Samples were incubated on ice for 45 min,
and then the erythrocytes were lysed and the preparation was suspended in
paraformaldehyde and sheath fluid. The monoclonal antibodies utilized for the
following cells are indicated in parentheses: lymphocytes (CD45) (KC56-fluo-
rescein isothiocyanate), monocytes (CD14) (MO2-RDI), and granulocytes
(CD11b) (forward and side scatter, no antibodies), T cells (CD3), B cells
(CD19), NK cells (CD56), T helper cells (CD4), T-cell suppressor/cytotoxic T
cells (CD8), T-cell suppressor inducer (CD4 plus CD45RA), T helper cell in-
ducer (CD4 plus CD29), and activated T cells (CD3 plus HLA-DR, DP, and
DQ).
Appropriate filter combinations were used to simultaneously measure emis-
sions from fluorescein isothiocyanate and phosphatidylethanolamine. Gates were
selected with forward and 90°-angle light scatter to select the cell population of
interest. A minimum of 1,000 cells were included in each analysis. The color
compensation was performed by examining the percentage of Fl1 (green) seen in
the Fl2 (red) and, conversely, the percentage of Fl2 seen in the Fl1 channel. For
this determination, CD4FITCI and CD8RD were utilized as staining control
lymphocytes. Routinely, F11 had 0 to 3% of F12, while F12 had 3 to 5% of F11.
Both the percentage and the absolute number of each cell type were determined.
Mitogen-induced lymphocyte stimulation. Mitogen-induced lymphocyte stim-
ulation was performed according to the techniques modified by Keller et al. (19)
with dose-response curves for concanavalin A (ConA; Calbiochem, San Diego,
Calif.), phytohemagglutinin (PHA; Welcome Reagents, Ltd., Beckenham, En-
gland), and pokeweed mitogen (PWM; GIBCO BRL Products). The doses per
well were as follows: for ConA, 3.75, 7.5, and 15 mg/well; for PHA, 0.05, 0.25, and
2.0 mg/well; and for PWM, 0.25, 0.5, and 5.0 mg/well. All lymphocyte stimulation
data were expressed as counts per minute in the stimulated cultures minus the
counts per minute in the unstimulated cultures. To approximate homogeneity of
variance, all counts were log transformed. The mean of the two higher doses was
utilized as a single dependent variable for regression analyses. (The lowest dose
was used to establish a basal response for the dose-response curve.)
NK cell activity. NK cell activity was assessed with K562 target tumor cells
according to standard methods modified by Georgescu and Keller (12, 13).
Target K562 cells were maintained by passaging every 2 to 3 days. Cell viability
was always .98%. On the day of assay, target cells are collected, washed, and
labeled with 500 mCi of
51
Crfor2hat37°C. Target cells are then washed three
times, and 10
4
cells are plated in microtiter plate wells. The NK cells are isolated
from the whole blood as described above and prepared in 3 dilutions in RPMI
with 15% normal human serum. The final concentrations of NK cells are 25 3
10
4
,503 10
4
, and 100 3 10
4
. This provides three effector-to-target ratios (25:1,
50:1, and 100:1). The mixture of target cells and NK cells is then incubated for
4 h at 37°C. The microtiter plates are then centrifuged, and the supernatant from
each well is assessed for
51
Cr activity. The NK data is presented as percentage of
specific cytotoxicity (see Data Analyses).
Granulocyte activity. Granulocyte activity was assessed by examining both the
phagocytic and killing ability of granulocytes according to the method described
by Weir (41), with the following modifications. After separation of the lympho-
cytes from the peripheral blood by a Ficoll-Hypaque gradient, the granulocytes
were separated from the erythrocytes by a percoll gradient. The granulocytes
were incubated with opsonized Staphylococcus aureus for 20 min. The incubation
mixture was centrifuged, and the unphagocytized S. aureus was washed out with
cold RPMI medium. Granulocytes were resuspended in RPMI, and aliquots
were prepared for further processing. One aliquot was processed immediately to
assess the phagocytic ability of the granulocytes. Two aliquots were incubated at
37°C to assess killing ability, and two aliquots were incubated on ice as controls.
At time 0, 1-h, and 2-h postincubation with S. aureus, the granulocytes were lysed
with 0.5% bovine serum albumin in distilled water and plated on blood agar
plates. The plates were incubated at 37°C for 24 h. The number of colonies was
counted; each represents an ingested S. aureus bacterium. Differences between
the numbers of colonies at 37 and 0°C represent specific killing of bacteria.
Data analyses. Data were first examined descriptively for distribution, means,
and variance. Multivariate models, defined a priori, were tested by regression
analyses. For each analysis of the enumerative measures, we examined the
contribution of age, gender, or race, controlling for the other two variables and
for the total leukocyte count (WBC) (simultaneous, not hierarchical), with num-
ber of leukocytes covaried. For the mitogen response, NK cell activity, and
granulocyte activity, WBC was not included in the model. All tests were two-
tailed.
The mean of the highest two responses to each of the mitogens and the mean
of the two highest effector-to-target killing ratios for NK cell activity were
utilized to form dependent variables for these regression analyses. Also, while we
examined the NK cell activity results both as specific killing and as the number
of lytic units, we have presented the data as percentage of killing for several
reasons. First, the use of specific killing was closer to the raw data and less
manipulated and allows direct inspection of the levels of specific cytotoxicity.
Secondly, not all of our data fits the assumptions required for lytic unit trans-
formation and requires either the dropping of particular data points, the drop-
ping of particular cases, or the truncation of the data. None of the options seems
preferable to the presentation of the data as percentage of killing.
The granulocyte activity assay was initiated in the latter part of the study.
However, sufficient numbers of adolescents were studied to permit analysis of
this data (n 5 96).
RESULTS
Demographics. As shown in Table 1, there were 100 males
and 106 females in this sample of healthy subjects (n 5 206).
The sample was comprised of 177 (83 female) African-Amer-
icans and 29 (17 female) Latinos/Latinas. The mean age 6
standard deviation for the entire sample was 15.8 6 1.7 years.
The mean age of the males was 15.75 6 1.81, and that of the
females was 15.83 6 1.59. The age distribution is presented as
a function of gender in Fig. 1. The age, gender, and race
distributions provided sufficient power to allow us to meaning-
fully assess their contributions to the variance of the immune
measures.
Immune measures. The findings for the entire sample are
presented in Table 2 and Fig. 2 through 4. The enumerative
measures are presented in Table 2 both as absolute number of
cells present and as a percentage of the total WBC. The dose-
response curves for each of the three mitogens are shown in
Fig. 2. In Fig. 3, the dose-response curve of NK cell activity at
the three effector-to-target ratios utilized is shown. In Fig. 4,
the killing of S. aureus (at 37°C and on ice) by granulocytes is
presented.
FIG. 1. Age distribution as a function of gender (n 5 206).
TABLE 1. Demographic data for subjects in this study
Characteristic
Female
(n 5 106)
Male
(n 5 100)
Total sample
(n 5 206)
African-American 83 94 177
Latino/Latina 17 12 29
Age (mean 6 SD) (yr) 15.8 6 1.6 15.8 6 1.8 15.8 6 1.7
106 BARTLETT ET AL. C
LIN.DIAGN.LAB.IMMUNOL.
Effects of age, gender, and race. We examined the contribu-
tions of age, gender, and race on the various immune param-
eters.
Age effects. (i) Cell phenotypes. WBC was positively corre-
lated with age (partial r 5 0.25, P , 0.001) (Fig. 5). Age also
contributed to the percentage of CD29
1
cells (F 5 3.25, P ,
0.002) (Fig. 6), with older subjects having a higher percentage;
to the number (F 5 2.31, P , 0.03) and percentage (F 5 2.14,
P , 0.04) of B cells, with younger subjects having higher
values; and to the percentage of NK cells (F 5 2.34, P , 0.03),
with older subjects having higher percentages.
(ii) Mitogen-induced lymphocyte stimulation. For the pro-
liferation assays, there were no significant effects of age on the
mean lymphocyte responses to the mitogens ConA (F 5 0.56,
P . 0.10), PWM (F 5 1.17, P . 0.10), or PHA (F 5 1.60, P .
0.10).
(iii) NK cell activity. There were no relationships between
age and mean NK cell cytotoxicity (F 5 0.62, P . 0.10). When
the number of NK cells was also controlled, this relationship
did not change substantively.
(iv) Granulocyte activity. This assay was initiated in the
latter part of the study. However, sufficient numbers of ado-
lescents were studied to permit the analysis of this data (n 5
96). As seen in Fig. 7, there was a significant decrease in the
phagocytosis of S. aureus with increasing age (F 5 3.33, P ,
0.01). In addition, after peaking when the subjects were age 14,
FIG. 2. Dose-response curves of the lymphocytes to each of the three mito-
gens (ConA, PHA, and PWM). Data has been log transformed and is presented
as the mean 6 the standard error of the mean (n 5 206).
FIG. 3. Dose-response curves of the NK cell killing at the three effector-to-
target ratios. Data is presented as the mean 6 the standard error of the mean
(n 5 206).
FIG. 4. Killing of S. aureus by polymorphonuclear granulocytes. Data is pre-
sented as the mean 6 the standard error of the mean (n 5 206).
TABLE 2. Totals for peripheral leukocyte measures for
entire sample
Variable
No. of cells (10
6
)/ml
(mean 6 SD)
% Leukocytes
(mean 6 SD)
Leukocytes 7.11 6 2.82
Lymphocytes 1.57 6 0.54 37.25 6 9.71
Granulocytes 2.63 6 1.18 55.39 6 10.35
Monocytes 0.29 6 0.12 6.68 6 2.38
T lymphocytes 1.22 6 0.46 76.55 6 8.48
B lymphocytes 0.23 6 0.13 15.29 6 5.94
CD4
1
lymphocytes 0.69 6 0.27 42.98 6 7.38
CD8
1
lymphocytes 0.33 6 0.16 20.95 6 5.46
Helper-to-suppressor ratio 2.31 6 0.07
CD29
1
lymphocytes (inducers
of help)
0.29 6 0.15 17.44 6 6.63
CD45RA
1
lymphocytes
(inducers of suppression)
0.34 6 0.17 21.12 6 6.67
CD56
1
lymphocytes (NK cells) 0.07 6 0.08 4.19 6 4.32
HLA-DR
1
lymphocytes
(activated T cells)
0.10 6 0.06 5.87 6 3.79
V
OL. 5, 1998 IMMUNE FUNCTION IN HEALTHY ADOLESCENTS 107
the percentage of bacteria killed at1h(F 5 2.13, P , 0.04) and
2h(F 5 2.95, P , 0.005) declined with age (Fig. 8).
Gender effects. (i) Cell phenotypes. As shown in Table 3, no
difference in total WBC or in numbers of lymphocytes, gran-
ulocytes, monocytes, or NK cells was found between males and
females. There was a significantly lower percentage (but not
number) of T cells in males than in females (F 5 5.85, P ,
0.0001). The number of B cells (F 5 3.43, P , 0.0009) was
higher in males, as was the percentage (F 5 2.14, P , 0.04).
There were significantly lower numbers of CD4
1
cells in the
male adolescents than in the female adolescents (F 5 2.24, P ,
0.03). Similarly, there was a lower percentage of CD4
1
cells
among males than among females (F 5 5.85, P , 0.0001).
Additionally, the percentage but not the number of CD29
1
cells (inducers of help) was lower among males than among
females (F 5 2.54, P , 0.02). Further, the helper-to-suppres-
sor ratio was higher in females than in males (F 5 2.44, P ,
0.02). No other differences in numbers or percentages of cells
were found.
(ii) Mitogen-induced lymphocyte stimulation. For the pro-
liferation assays, there were no significant effects of gender on
the mean lymphocyte responses to ConA (F 5 0.40, P . 0.10),
PWM (F 5 1.50, P . 0.10), or PHA (F 5 1.27, P . 0.10).
FIG. 5. WBC (mean 6 standard error) as a function of age; partial r, 0.25; P,
,0.001.
FIG. 6. Percentage of CD29
1
(inducer of help) as a function of age (mean 6
standard error); partial r, 0.31, P, 0.001.
FIG. 7. Number of S. aureus cells phagocytized as a function of age (P ,
0.04). Data is presented as the mean 6 standard error of the mean (n 5 206).
The 12- and 13-year-old subjects were combined due to the small number of
subjects in these two groups.
FIG. 8. The percentage of S. aureus cells killed as a function of age (P ,
0.01). The data is presented as the mean 6 standard error of the mean (n 5 206).
The 12- and 13-year-old subjects were combined due to the small number of
subjects in these two groups.
108 BARTLETT ET AL. CLIN.DIAGN.LAB.IMMUNOL.
(iii) NK cell activity. There was no significant relationship
between gender and mean NK cell cytotoxicity (F 5 1.08, P .
0.10). When number of NK cells was controlled, these results
were not altered (F 5 0.82, P . 0.10).
(iv) Granulocyte activity. There were no gender-based dif-
ferences in granulocyte phagocytosis (F 5 0.74, P . 0.10) or
killing activity at 1 (F 5 0.15, P . 0.10) or at2h(F 5 0.78, P .
0.10) of incubation.
Race effects. (i) Cell phenotypes. The associations between
race and cell numbers are presented in Table 4. WBC differed
with race, being lower for African-Americans than for Latinos/
Latinas (F 5 3.85, P , 0.0002). African-Americans had a lower
number of granulocytes than Latinos/Latinas (F 5 2.02, P ,
0.05) and tended to have a lower percentage of granulocytes
(F 5 1.72, P 5 0.09). African-Americans also had a lower
percentage (F 5 2.10, P , 0.04) but not a lower number (F 5
1.48, P . 0.10) of HLA-DR
1
lymphocytes (activated T cells)
than did Hispanics.
(ii) Mitogen-induced lymphocyte stimulation. For the pro-
liferation assays, there were no significant effects of race on the
mean lymphocyte responses to ConA (F 5 0.22, P . 0.10),
PHA (F 5 1.51, P . 0.10), or PWM (F 5 1.27, P . 0.10).
However, for PWM, African-American adolescents differed
from Latino/Latina youth in their dose-response curve (F 5
9.39, df 5 2 and 320, P , 0.0001) (Fig. 9). While the African-
Americans seemed to show higher proliferative response at the
lowest dose, the Latino/Latina adolescents showed higher pro-
liferation at the two higher doses.
(iii) NK cell activity. There were no relationships between
race and NK cell cytotoxicity (F 5 0.51, P . 0.10). With
number of NK cells also in the model, the relationship re-
mained nonsignificant.
(iv) Granulocyte activity. As seen in Fig. 10, race affected
granulocytic activity. Hispanic adolescents showed a higher
percentage of killing than did African-American adolescents at
1h(F 5 2.32, P , 0.03) and at2h(F 5 1.87, P , 0.07)
incubation.
Substance use effects. As adolescence is a time of psycho-
social as well as physical change, with frequent experimenta-
tion, including substance use, which can affect immunity, we
did preliminary analyses to assess possible effects of alcohol
and drug use on the immune parameters herein. Behavioral
data consisted of a self-report of alcohol and drug use during
the day and week preceding venipuncture and a self-report of
the average number of cigarette packs the subject smoked
during the past year. As substance abuse was an exclusion
criterion, and as these students were either in school or actively
seeking health care, the incidence of substance use was quite
TABLE 3. Peripheral blood leukocyte measures by sex
Variable
No. of cells (10
6
)/ml (mean 6 SD) Mean % leukocytes 6 SD
Male
subjects
Female
subjects
FPMale subjects
Female
subjects
FP
Leukocytes 6.92 6 2.86 7.28 6 2.80 1.44 .0.10
Lymphocytes 1.55 6 0.52 1.58 6 0.53 0.83 .0.10 38.45 6 10.11 36.20 6 9.45 1.66 .0.10
Granulocytes 4.25 6 1.91 4.54 6 2.01 1.45 .0.10 54.05 6 10.56 56.35 6 10.30 1.78 0.08
Monocytes 0.51 6 0.23 0.49 6 0.21 1.20 .0.10 6.87 6 2.07 6.71 6 2.72 1.00 .0.10
T lymphocytes 1.17 6 0.44 1.27 6 0.45 0.77 .0.10 73.68 6 9.01 79.50 6 6.74 5.08 0.0001
B lymphocytes 0.26 6 0.14 0.21 6 0.11 3.43 0.0009 16.83 6 6.54 13.71 6 5.01 2.14 0.04
CD4
1
lymphocytes 0.63 6 0.25 0.74 6 0.27 2.24 0.03 40.16 6 7.09 45.64 6 6.71 5.85 0.0001
CD8
1
lymphocytes 0.33 6 0.16 0.32 6 0.14 0.75 .0.10 20.78 6 5.31 20.88 6 5.53 0.23 .0.10
Helper-to-suppressor ratio 2.12 6 0.84 2.47 6 0.93 2.44 0.02
CD29
1
lymphocytes (inducers of help) 0.27 6 0.15 0.30 6 0.14 1.17 .0.10 16.26 6 6.76 18.86 6 6.13 2.54 0.02
CD45RA
1
lymphocytes (inducers of
suppression)
0.33 6 0.17 0.35 6 0.17 0.60 .0.10 20.30 6 6.65 21.88 6 6.63 1.34 .0.10
CD56
1
lymphocytes (NK cells) 0.08 6 0.10 0.06 6 0.06 1.56 .0.10 4.81 6 5.12 3.73 6 3.56 1.72 0.09
HLA-DR
1
lymphocytes (activated T cells) 0.11 6 0.07 0.09 6 0.06 1.79 0.08 6.47 6 4.48 5.50 6 3.29 1.48 .0.10
TABLE 4. Peripheral leukocyte measures by race
Variable
No. of cells (10
6
)/ml (mean 6 SD) Mean % leukocytes 6 SD
African-
American
subjects
Latino/Latina
subjects
FP
African-
American
subjects
Latino/Latina
subjects
FP
Leukocytes 6.75 6 2.71 9.07 6 2.69 3.85 0.0002
Lymphocytes 1.54 6 0.54 1.70 6 0.46 1.71 0.09 38.23 6 9.80 32.22 6 8.28 1.61 .0.10
Granulocytes 4.12 6 1.84 5.78 6 2.01 2.02 0.05 54.14 6 10.43 61.07 6 8.63 1.72 0.09
Monocytes 0.48 6 0.19 0.58 6 0.32 0.23 .0.10 6.90 6 2.44 6.18 6 2.39 0.48 .0.10
T lymphocytes 1.20 6 0.45 1.34 6 0.39 1.20 .0.10 76.46 6 8.46 78.03 6 8.16 1.10 .0.10
B lymphocytes 0.23 6 0.12 0.23 6 0.13 2.33 0.03 15.39 6 5.82 14.18 6 6.89 1.52 .0.10
CD4
1
lymphocytes 0.67 6 0.26 0.75 6 0.29 1.01 .0.10 43.02 6 7.25 43.09 6 8.31 0.35 .0.10
CD8
1
lymphocytes 0.32 6 0.16 0.35 6 0.11 1.21 .0.10 20.84 6 5.43 20.80 6 5.43 0.57 .0.10
Helper-to-suppressor ratio 2.31 6 0.84 2.32 6 1.18 0.44 .0.10
CD29
1
lymphocytes (inducers of help) 0.27 6 0.14 0.35 6 0.16 0.19 .0.10 17.17 6 6.63 19.86 6 5.75 1.38 .0.10
CD45RA
1
lymphocytes (inducers of suppression) 0.33 6 0.15 0.39 6 0.21 0.07 .0.10 20.90 6 6.33 22.29 6 8.23 0.90 .0.10
CD56
1
lymphocytes (NK cells) 0.06 6 0.06 0.11 6 0.14 1.79 0.08 4.06 6 4.41 5.30 6 4.27 0.70 .0.10
HLA-DR
1
lymphocytes (activated T cells) 0.09 6 0.06 0.13 6 0.06 1.48 .0.10 5.62 6 3.95 7.82 6 3.23 2.10 0.04
V
OL. 5, 1998 IMMUNE FUNCTION IN HEALTHY ADOLESCENTS 109
low in our sample. No subject had used alcohol or drugs in the
24-h period prior to venipuncture. In the preceding week, 8%
of the subjects had used alcohol, varying from one to two
glasses of wine or hard liquor to 1 to 40 beers (one subject had
had 40 beers more than 24 h previously, while the next highest
number of beers that had been drunk that week was 4). The
only drug the subjects reported using was marijuana, with only
one subject reporting this in the week prior to venipuncture.
No other substance use (except that of cigarettes) was reported
for the week.
The only immune measure affected by any of the reported
substance use was the killing of S. aureus, which was inversely
associated with alcohol use during the week prior to the veni-
puncture (data not shown).
DISCUSSION
This study of immunity represents the largest reported sam-
ple of normal healthy adolescents to date. This data demon-
strates differences in total WBC related to age and race; dif-
ferences in lymphocyte subtype counts related to gender, race,
and age; differences in PWM response related to race; differ-
ences in granulocyte phagocytic ability related to age; and
differences in granulocyte bactericidal activity related to both
age and race. These findings contribute to the literature con-
cerning normative data on adolescent immunology. Further,
these findings provide information on enumerative and func-
tional measures of both lymphoid and myeloid cells as they
relate to age, gender, and race. We found almost no effects of
alcohol or tobacco (when reported use was minimal) on im-
munity in these healthy adolescents. This data provides impor-
tant descriptive information on adolescent minority popula-
tions who are represented only minimally in the normative
data available to date. This data will be a useful measure of
comparison when minority adolescents with immune-related
problems or disease are to be assessed.
This data expands upon the work of Tollerud and coworkers
(40), who assessed lymphocyte subset numbers in 112 healthy,
predominantly Caucasian adolescents (mean age, 15.4 6 1.9;
range, 12 to 19 years). Our mean age and age range were
almost identical to those of Tollerud et al. (15.8 6 1.7 and 12
to 18 years). The populations investigated were different as far
as racial mix. With respect to the immunologic measures in-
vestigated, we included functional as well as enumerative mea-
sures. However, our enumerative findings are similar to those
of Tollerud et al. With regard to gender differences, in both
studies there were greater numbers of B cells (in the peripheral
circulation) in males. Both studies also suggest racial differ-
ences in enumerative measures. Tollerud and coworkers re-
ported increased numbers of B cells in black males compared
to those in Caucasians, similar to our finding of higher num-
bers of B cells in African-Americans compared to those in
Latinos/Latinas. Further, Tollerud et al. reported that their
older adolescent female subjects had a higher proportion of
CD4
1
cells than the older adolescent males (ages 17 to 19),
similar to our finding that females (12 to 18 years of age) had
a greater number and percentage of CD4
1
cells than males.
There were also differences between the present study and
that of Tollerud et al. While Tollerud et al. found gender
differences in CD8
1
cell counts, we did not (nor did we find
racial differences in CD8
1
counts). Further, Tollerud and co-
workers found that the CD4
1
-to-CD8
1
ratio was higher in
males, while we found that it was higher in females. Addition-
ally, the data of Tollerud et al. suggest that this ratio was higher
in blacks than in Caucasians, while we found no differences
between blacks and Hispanics. These differences may reflect
gender-race interaction effects which might be quite different
due to the difference in racial composition between the two
samples.
Studies of populations much more diverse than that in the
present study also report enumerative differences related to
age. For example, Comans-Bitter and colleagues (8) also re-
ported on cell numbers in subjects from infancy to adulthood.
These researchers’ sample included 23 children from ages 10 to
16 years as part of a study of age-related differences in cell
counts and percentages. While the subjects studied were not
comparable to ours, Comans-Bitter et al. reported that the
percentage of NK cells increases with age, while the actual
FIG. 9. Lymphocyte proliferation in response to PWM by race (P , 0.0001).
Data has been log transformed and is presented as the mean 6 standard error of
the mean (n 5 206).
FIG. 10. The percentage of S. aureus cells killed as a function of race (P ,
0.05). The data is presented as the mean 6 standard error of the mean (n 5 206).
110 BARTLETT ET AL. CLIN.DIAGN.LAB.IMMUNOL.
number of cells appears to remain stable. In our more re-
stricted age range, we found a similar trend for an increased
percentage but not an increased number of NK cells. While
Comans-Bitter et al. reported no statistical findings, their ob-
servations that age may affect the percentage but not the num-
ber of NK cells are similar to ours in that numbers and/or
percentages of lymphocytes can vary with age independently of
each other.
Ihara and colleagues (15) assessed polymorphonuclear leu-
kocyte functioning (H
2
O
2
generation, which suggests killing
activity) in 58 children and adolescents and reported increased
H
2
O
2
production by polymorphonuclear leukocytes exposed to
S. aureus or Escherichia coli with increased age (6 months to 15
years). Further, Ihara et al. found that adolescents’ (ages 10 to
15 years) granulocyte function (H
2
O
2
production) was similar
to that of adults, while children’s H
2
O
2
production was lower.
We found that bacterial killing peaked when the subjects were
14 years old and then declined with age to values lower than
those seen in the 12- to 13-year-old adolescents by age 18. The
results from our study seem different in that we would predict
decreased H
2
O
2
production in adults compared to early ado-
lescents, while Ihara et al. found that H
2
O
2
production in
young adolescents and adults was quite similar. These differ-
ences may relate to the differences in study design, making the
comparability of these two data sets difficult to assess (Ihara
and colleagues measured hydrogen peroxide generation, an
indirect measure of bactericidal activity, while we measured
directly the number of bacteria that were ingested and killed).
Further, the data presented by Ihara encompassed a much
greater age range, with infants, prepubescent children, and
pubescent or postpubescent adolescents, while our only sub-
jects were postpubescent adolescents. However, despite these
large methodological differences, both studies support the hy-
pothesis that granulocyte function changes with age in young
people.
Having demonstrated age-, gender-, and race-related differ-
ences in immunity during adolescence, one may speculate as to
mechanisms possibly involved. Hormonal factors, especially
those present during adolescence, may have influenced our
results and offer venues for future investigation. For example,
hormonal changes associated with growth and development
may affect immunity. Indeed, peripheral blood lymphocytes
have receptors for hormones such as growth hormone (GH)
(20), GH releasing factor (GRF), and somatostatin (5), and
some peripheral blood lymphocytes have been found to pro-
duce GH, somatostatin, and a GRF-like peptide (5). Some of
the age-related differences we found might be related to GH or
GRF, but it is possible that the majority of the 12- to 18-year-
old subjects we studied were actively growing with similar lev-
els of growth hormones.
The possibility that other growth-related hormonal factors
account for some of the age-related differences we describe is
suggested by the literature concerning the secretion of insulin-
like growth factor (21), which is produced in response to se-
cretion of GH. The secretion of insulin-like growth factor
increases in both male and female adolescents, peaks higher
and earlier in girls, and decreases during the latter half of
adolescence to adult levels (1). This finding may help explain
the age differences and suggests that age effects may be sub-
sumed in gender findings, as girls have the most rapid growth
during early puberty (before the onset of menses), while boys
grow more during midpuberty (25). We undertook age by
gender interaction tests to explore this and found only one
significant measure (age by sex test for B-cell numbers, P ,
0.04). Future studies comparing adolescents to same-sex fully
mature adults may further delineate this issue.
Sex steroids, which also change significantly during pubertal
development and continue to fluctuate in diurnal or monthly
rhythms, may also have influenced our findings. Estrogen
and/or progesterone may affect immunity directly or indirectly
(2, 10, 23, 24, 27, 30, 32, 33, 37). Enumerative (34, 38) and
functional myeloid (16, 17, 29, 36) and lymphoid (7, 28, 37, 39,
42) measures have been reported to be affected by sex steroids.
Enumerative immune measures may be affected by sex ste-
roids. Casson and colleagues (6) reported that the percentage
of CD4
1
cells was decreased, while that of NK cells was in-
creased, when postmenopausal women were treated with de-
hydroepiandrosterone. Kiess and coworkers (22) reported
lower percentages of CD4
1
cells in untreated males with hy-
pogonadism compared to those in normal healthy men and
those in subjects with treated hypogonadism, which resulted in
normal testosterone levels. Our finding of lower numbers of
CD4
1
cells in males is consistent with Casson and coworkers’
(6) findings suggesting that circulating androgens (in post-
menopausal women) are associated with decreased numbers of
CD4
1
cells.
Concerning sex steroid effects on mitogen stimulation, Yron
and colleagues (42) found that neither 17 beta-estradiol nor
progesterone altered the response to ConA. We found no
gender differences in T- or B-cell response to mitogen stimu-
lation (PHA, PWM, or ConA). Therefore, our findings are
consistent with those of Yron et al. and suggest that response
to mitogen stimulation is not significantly affected by gender
differences in gonadal hormones.
Contradictory findings for effects of sex steroids on NK ac-
tivity have been reported. For example, Sorachi and coworkers
(37) reported that 17 beta-estradiol (E2) enhances NK activity,
while progesterone and testosterone do not. Liu and Hansen
(26) reported that NK cytotoxic activity was inhibited by pro-
gesterone. Mandler and coworkers (27) reported that NK cell
depolarization is affected by progesterone but not by estrogen.
Contrarily, Callewaert and coworkers (4) reported that NK cell
activity was not affected by high concentrations in vitro of
testosterone, progesterone, or estradiol. We did not find any
gender (or age 3 sex)-related differences in NK cell activity,
and therefore our findings are more in keeping with the im-
plications of the results reported by Callewaert and colleagues.
However, we did not control for stage of menstrual cycle,
which at peaks of estrogen or progesterone might have yielded
very different results.
In addition to age and gender effects, we found significant
racial differences in some enumerative measures and in gran-
ulocyte function (African-Americans compared to Latino/
Latina youth). Tollerud and coworkers also found racial dif-
ferences in enumerative measures (percentage of HLA-DR
1
)
(Caucasians versus blacks). Ihara and colleagues (15) did not
indicate their subjects’ race, but presumptively their sample
was all Japanese, thereby precluding any investigation of racial
differences. The biological basis of racial differences remains to
be explored. However, this data suggests the existence of racial
differences in immunity which must be addressed whenever
immune-related disease processes are investigated.
The potential for selection bias in this data must be ad-
dressed. The subjects in the present study were recruited from
an inner-city high school and an inner-city adolescent general
medical clinic. Students were members of randomly selected
10th grade English classes, and greater than 90% of the stu-
dents in each targeted class agreed to participate. Similarly, in
the adolescent clinic, consecutive patients were approached for
participation in the study, and greater than 80% of these ad-
olescents agreed to participate. Results from studies with high
school dropouts or children with academic problems requiring
VOL. 5, 1998 IMMUNE FUNCTION IN HEALTHY ADOLESCENTS 111
special education services might be dissimilar to those of the
present study.
Concerning the physical well-being of the subjects in the
present study, all were categorized as healthy by physicians,
based on both history and physical examination. Since these
subjects were part of a larger study on behavioral and biolog-
ical AIDS risk factors, we also collected a large, intimate be-
havioral and psychological data set on the same day as veni-
puncture. The subjects had no reason to misrepresent their
health, and they were asked much more personal information
than when they had most recently been ill. The time frame for
recall of intercurrent health problems was short (2 weeks) and
thus should not have represented a problem in recall. The
random selection of the subjects, the physical assessment by
physicians, the collection of intimate psychobehavioral data
and venipuncture on the same day, as well as the immediate
processing of the blood for each of the multiple immune assays
were important factors in assuring the quality of this normative
data.
The effects of demographic factors on a wide range of im-
munological variables demonstrate the importance of having
normative data representative of particular patient popula-
tions. Even though our subjects were randomly sampled from
the same general population, there were marked immunolog-
ical differences in subgroups defined by age, gender, and race.
If the stability of these factors over time is addressed in these
types of studies, researchers will have an even clearer picture
of the normative values of immunological functioning in ado-
lescents.
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