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All Graduate Theses and Dissertations Graduate Studies
5-1985
Cellular Immunity in Children with Down Syndrome (Trisomy-21)
Roger Lee Noble Utah State University
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This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. CELLULAR IHHUNITY IN CHILDREN WITH
DOWN SYNDROME (TRISOMY-2 1)
by
Roger Lee Noble
A dissertation submitted in partial f•tlfillment of t he requirements for the degree
of
DOCTOR OF PHILOSOPHY
in
Biology ( Hlc robi ology / Immunology)
Approved :
UTAH STATE UNIVERSITY Logan , Utah
1985 ii
ACKNOWLEDGEMENTS
I must thank Dr. Reed P. Warren, my major professor and mentor for his enthusiasm for immunology and his influence and support as I have pursued my goals. I appreciate the members of my committee,
Dr. Frederick J. Post, Dr. John R. Simmons, Dr. Stanley D. Allen and
Dr. Mark C. Healey for their friendship and willingness to help.
I am grate~1l to Dr. Donald V. Sisson for his valuable assistance with matters statistical. Other friends who have been especially supportive, Nancy Pace and Dr. Nadine Margaretten, I thank for their technical support and incite.
I express gratitude to my dear wife Nan for her patience and the monetary and moral support which I desperately needed as I worked on this project. To my parents I owe credit for the desire which I have had to pnrsne excellence in my education. I also humbly express thanks to God for the ability to pursue this endeavor and for creating the world around and in us for onr discovery.
Roger· Lee Noble iii
TABLE OF CONTENTS
Page
ACKNOWL EIX;EMENTS ii
LIST OF TABLES. v
LIST OF FIGURES vi
LIST OF ABBREVIATIONS vii
ABSTRACT. viii
INTRODUCTION
REVIEW OF LITERATURE. 4
Down Syndrome 4 Effect of Age in Down Syndrome. 5 Immunity and Down Syndrome • 6
Blood Cell Populations. 7 The Phagocytic Sys tern • 8 The Antibody...... ,diated System. 8 The Cell-mediated System • 9 Zinc and Immmity in Down Syndrome. 13 Gene-loci on Chromosome-21 14 Imm1ni ty in Down Syndrome Children. 15
MATERIALS AND METHODS 17
Down Syndrome Subjects and Controls 17 Collection and Separation of Blood Samples. 17 Hematocrits, Total White Cell Connts, and Differential Counts 18 SRBC-roset ting With and Without AET-treatm:.n t. 18 Enumeration of T Lymphocyte Subsets • 20 Mitogen-stimulated Lymphocyte Proliferation 22 Interleukin-2 (IL-2) Production and Assay of IL-2 22 Assay of Natural Killer Cell Activity and Interferon Augmentation 24 Assay of Plasma Zinc 26 Statistical Methods 27
... iv
RESULTS • 29
Enumeration of Various Cell Populations in the Blood 29 Proliferative Response to Mitogens 37 Interleukin-2 (IL-2) Production 42 Natnral Killer Cell Activity and the Response of Nab1ral Killer Cells to Interferon. 42 Plasma Zinc Levels. 46
DISCUSSION • 53
CONCLUSIONS. 63
LITERATURE CITED 65
VITA • 72 v
LIST OF TABLES
Table Page
1. Enumeration of blood cell subsets in young children with Down syndrome (DS) 30
2. Chi-s~•are analysis of data from differential blood cell counts in young children with Down syndrome. 31
3. Partitioning of chi-square analysis of data from differential blood cell counts in young children with Down syndrome • 32
4. Comparison of two techniques for enumerating rosette-forming cells in young Down syndrome children. 34
5. Comparison of complement-dependent cytoxicity (CDC) and the Quantigen kit as methods for determining OKT4+ and OKT8+ cells and T4:T8 ratios 38
6. Interleukin-2 production by stimulated lymphocytes from young children with Down syndrome (DS). 44
7. Interferon augmentation of natural killer activity in young children with Down syndrome • 47
8. Comparison of regression slopes for baseline and interferon-augmented natural killer cell activity of cells from Down syndrome and control subjects by analysis of covariance. 49
9. Comparison of regression slopes for baseline and interferon-augmented natural killer cell activity of cells from Down syndrome subjects by analysis of covariance. 50
10. Comparison of regression slopes for interferon augmented natural killer cell activity of cells from Down syndrome and control subjects by analysis of covariance. 51 vi
LIST OF FIGURES
Figure Page
l. Depressed ratio ofT cell subsets (OKT4+:0KT8+) in young children with Down syndrome • 35
2 . The number of circulating OKT4+ cells in ymng children with Down syndrome (DS) is significantly depressed 36
3. Depressed proliferative respons3 to phytohemagglutinin (PHA), as c011nts per minute of H-thymidine uptake by lymphocytes from young children with Down syndrome 39 3 4. Concanavalin A (Con A)-stimulated H-thymidine up take by lymphocytes from yonng Down syndrome and control children 40
5. Stimulation of lymphocyte proliferation by pokeweed 3 mitogen, measured as counts per minute of H-thymi.dine uptake, in young children with Down syndrome 41
6. Probit analysis of the sigmoidal response cnrve from assay of a laboratory standard solution (supernatant from Concanavalin A- s tim1lated rat splenocytes) of interleukin-2 (IL-2) 43
· 7. ~seline natural killer cell activity, induction of Cr -release from K562 cells by PBMC from young fuwn syndrome children and age""111atched controls 45
8 . Regression lines to assay the effect of increasing effector to target cell ratios (E:T) and of interferon treatment on nab1ral killer activity in Down syndrome subjects. 48
9. Zinc levels in plasma from young children with fuwn syndrome (DS) 52 vii
LIST OF ABBREVIATIONS
Abbreviation Definition
AET 2-ami no-e thyl-i sothiou ronium bromide B • humoral immunity-mediating CDC complement-dependent cytotoxicity Con A concanavalin A ns. Down syndrome, trisomy-21 FBS fetal bovine serum IFN interferon Ig. immunoglobulin IgA immu noglobulin- A IgG immunoglobuli n-G IgM immunoglobulin-M IL-2. interleukin- 2, T cell growth factor 2- ME. 2-mercapto- ethanol NK . • natural killer PBMC. peripheral blood monom•clear cells PBS phosphate ~•ffered sali~e PHA p hy t ohemagg lu ti ni n PMA 4-phorbol 12-myris tate 13-acetate PWM pokeweed mitogen RFC SRBC rosette-forming cells S.D •• standard deviation s.e .m .. standard error of the mean SRBC. sheep red blood cells T • • thy~• s-derived , bearing SRBC-R (except when otherwise defined) T4:T8 ratio of OKT4+ cells to OKT8+ cells WBC • white blood cells viii
ABSTRACT
Cellular IImmnity in Children with
Down Syndrome (Trisomy-21)
by
Roger lee Noble, Doctor of Rlilosophy
Utah State Unive r sity, 1985
Major Professor: Dr. Reed P. Warren Department: Biology
Individuals with Down syndrome (US) suffer from increased incidence of respiratory infections and lymphoblastic leukemia, a nd a death rate
that is particularly high in the first 5 years of life. Rela t ively few s01dies have probed imm•ne parameters in ya•ng US children. Primary immtne defects in DS may be masked by a degree of imnntne maturity in adults , and hygienic factors may have an effect on imnune capability
throughout the years. A study of young children can give clearer evidence of the actual p r imary imm•ne defects in ns.
Blood samples were drawn from 20 DS children under 6 years old and from age~atched controls. Packed blood cell volume was measured, various blood cell snbpopnlations were enumerated and differential cotnts were perforned. Seve ral tests of cellular immtne function ~re performed and plasma zinc l evels were determined using atomic adsorption spe ctr opho tome try.
Elevated hematocrit levels were observed in the US group. White blood cell counts a nd proportions of rosette-forming cells were normal ix
in blood from DS children. Altered neutrophil and lymphocyte
proportions in DS samples resulted from a depressed nnmber of
circnl,ting lymphocytes in the these subjects. This indicated that T
cell numbers are low in DS. DS individuals had a low nnmber of
circulating OKT4+ T cells which resulted in significantly depressed
T4:T8 ratios. Cells from DS subjects exhibited a reduced proliferative
r e sponse to phytohemagglutinin; a low response to the optimal
concen tration of concanavalin A was seen with OS samples, but at
snboptimal doses the response was normal; suboptimal concentrations of
pokeweed mitogen elicited normal responses by cells from DS children.
Preliminary results suggest that interlenkin-2 (IL-2) production in yon ng children may correlate positively with age and that DS snbj ects may produce normal or elevated amounts of IL-2. This suggests that IL-2
receptor function may be defective in T cells from DS childre n. ns children had normal natural killer cell activity and cells from those children were no more sensitive to the augmenting effects of interferon-alpha than cells from control children. Plasma zinc levels in DS appeared to be normal.
These findings not only provide evidence that the primary immnne defect in DS involves low levels of T cells, but they show depressed number and function of helper T cells and suggest defective IL-2 receptor expression in bs.
(83 pages) INTRODUCTION
1 Down syndrome (DS) is a common cause of mental retardation in
humans resulting from an extra replicate of a portion or all of
chromosome 21 in the genome of the affected individual. The mechanism
leading to retardation is not understood. Approximately 0.1- 0.2% of all
babies born worldwide have DS (Wahrman and Fried 1970; Sever et al.
1970; Lott 1982). A unique set of characteristics and conditions,
mental, morphological and physiological, are observed in these
individuals. An unusually high incidence of respiratory infections in
patients with OS, as well as an elevated frequency of anti-thyroid
antibodies and increased risk of acute lymphobiastic leukemia (Scholl et
al. 1982; Fialkow !970), implicate the immune system and suggest defective immune function. Consequently, many reports of the number and
proportions of immunocompetent cells and the functional status of
cellular immunity and the phagocytic system in OS have been published.
However, the majority of those were studies of adults with DS, and
relatively few studies have dealt with young DS patients. Since the
increased rate of mortality is highest during the earliest years of life
(Wahrman and Fried !970), those earlier studies of older individuals were making observations in a select group of survivors.
The purpose of the study reported in this dissertation was to investigate immune parameters in young children to give a clearer view of the primary immune defects found in DS. The project involved
sampling blood from children 5 years old and younger who had OS,
1 A list of abbreviations can be fm1nd on pg. vii . assaying the samples for im~tne parameters and then comparing the
results with data derived by studying samples from healthy age-matched
controls .
When possible, the hematocrit was measured, a total white blood
cell (WBC) count was made, and a differential smear was prepared and
counted. Depending on the number of peripheral blood mononuclear cells
(PBMC) obtained from each individual, T cells were enumerated and the
proportions of T cell subsets (OKT4+ and OKT8+) determined.
Tests of several immune functions were performed to determine what
abnormalities might exist in children with DS. The proliferative
response of lymphocytes to phytohemagglutinin (PHA), concanavalin A (Con
A) a~d pokeweed mitogen (PWM) was assayed . Natural killer (NK) c~ll
activity was tested and the level of augmentation of the NK activity by
inte rferon (IFN) was investigated in order to determine the sensitivity
of these cells to IFN. This was of interest because the genetic determination of sensitivity to the effects of IFN has been assigned to
the distal end of the long arm of chromosome 21 (Epstein et al. 1980),
the chromosome which is trisomic in DS.
The trace element zinc is a necessary cofactor for many metalloenzymes, including several which are required for the synthesis of DNA and RNA. Such synthesis is crucial in the proliferative function of lymphoid cells. It has been shown that zinc deficiency has a profound influence on the thymus resulting in thymic involution, depressed DNA content and reduced levels of thymic hormone (Good 1981).
Also, zinc deficiency leads to alterations in thymus-mediated immune mechanisms such as depressed helper and killer T cell function (Good
1981). In DS, thymuses often are small, depleted of lymphocytes and 3 exhibit diminution of the cortex and a loss of corticomedullary demarcation (Levin et al. 1979). Because of the im1m1ne deficiency in OS and these histological abnormalities, plasma zinc levels were determined in this study. 4
REVIEW OF LITERATURE
Down Syndrome
DS was initially described by Seguin in 1846, and was characterized
more definitely in 1866 by Sir Langdon Down, as "mongolian idiocy". The
term "mongolism" has been all but dropped from common usage in favor of
"Down syndrome". About 95% of patients with DS are trisomic for
chromosome 21 (Lejeune et al. 1959), .tlile other DS patients have either
"translocation" or "mo saic" DS (National Institutes of Health 1979).
The subjects included in this sb•dy were of the trisomic type only.
Although known primarily as a condition of physical abnormalities
and profound mental retardation, DS also has a striking association with
other diseases which have resulted in increased rates of mortality
(Sever et al. 1970; Wahrman and Fried 1970; Deaton 1973; Seger et al.
1977). With the advent of antibiotic therapy, life expectancy for DS
individuals increased from about age 9 years in 1929 to about 35 years
of age in 1969 (Thase 19 82; Deaton 1973). Recently, it was reported
that an es t imated 50% of all people born with DS now live to middle-age
(Jones 1979). In spite of this increased survival time, the risks of infection and mortality remain elevated in these patients (Oster et al.
1975; Scholl et al. 19 82) . Respiratory infections and pneumonia occur in increased incidence and are the leading causes of death in all DS
patients (Siegel 1948a; Donner 1954; Oster et al. 1964; Sever et al.
1970; Kaldor and Pitt 1971; Deaton 1973; Oster et al. 1975; Scholl et
al. 1982). Lymphocy tic leukemia, of uncertain etiology, also occurs at 5
higher frequency in the DS population than i n the general population and
is another commo n cause of death (Krivit and Good 1956; Miller 1964;
Oster et al. 1975). Mortality caused by leukemia seems t o be especially
severe in the group of DS subjects ( 5 years old (Scholl et al. 1982 ) .
The other leading cause of death in DS, cardiovascular disease, is due
primarily to congenital anomalies (Scholl et al. 1982).
Chronic antigenemia for the hepatitis B virus surface antigen (also
known as the Australia antigen) is observed more frequently in
institutionalized DS patients than in their non-DS counterparts
(Blumberg et al. 1967 ; Sutnick et al. 1972; Smith et al. 1982).
However, no significant correlations between this condition and o ther
i~1ne abnormalities have been noted.
The above observations have led many researchers to s u spect that a
primary immunodeficiency exists in DS. Consequently, many studies of DS
individuals have probed immune parameters.
Effect of Age in Down Syndrome
Of the many studies of imrrn1ne functions in DS which have been
performed, the majority deal strictly with adults or disregard age
altogether. Only a few have specifically dealt with the young DS child.
It has been pointed out that "the age problem deserves emphasis"
(Walford 1982).
The death rate in DS is particularly high in subjects ( 5 years old
and in those over 40 years of age (Deaton 1973), and is highest during
the first year of life (Sever et al. 1970; Wahrman and Fried 1970).
Prior to the antibiotic era, about 75% of the DS patients died before puberty (Seger et al. 1977). Specifically, 0-4 year old DS children 6 have a 3-fold greater incidence of leukemia than expected (Krivit and
Good 1956) and mortality from leukemia is reported to be especially
severe in the< 5 year old age group (Scholl et al. 1982).
Differences between the immune capacity of OS adults and that of OS children, when they are compared to age-matched normal controls, are evident. Some studies which report these differences will be mentioned in the section below. Also, there are several im~1ne parameters for which no data from young OS children have been reported.
It is possible that DS subjects "grow on t of" some immune deficiencies, a concept which could account for the decreased mortality seen in middle-aged patients. This maturation of the OS immune system may mask primary immune defects. It has been suggested that environmental factors, particularly poor personal hygiene due to mental retardation and crowding of institutionalized patients, may lead to a heavy load of infections in early life, and that this may result in a
"stress--deficiency" of the cell-mediated immune system (Whittingham et al. 1977). Such age and environmental factors may cloud the issue in older OS subjects and make it difficult to distinguish primary immune defects caused by trisomy-21 alone. The current study of imnntne capacity in young OS children avoids selecting only those individuals who survived beyond the fifth year and more clearly identifies abnormalities caused primarily by the genetic defect of t risomy-21.
Immunity and Down Syndrome
Imm11ne factors and immunopotent cells are circulated via the peripheral blood. WBC are the cells most directly involved in im~tnity.
The various types of WBC perform extremely diverse functions in an 7
immune response. It is useful to think of the immune system as four
interelated subsystems: the phagocytic system, the antibody-mediated
system, cell-mediated immunity, and the complement system. All but the
complement system are under control of the thymus by means of thymic
"hormones" and factors, and by thymic influence on certain stem cells as
they mature and become T lymphocytes (T cells). The immune system
includes populations of specialized WBC which maintain surveillance and
detect, respond to, and neutralize in various ways foreign substances
and abnormal conditions which may arise in the body. Measurable
indicators of the competence of immunity include observations of cell
morphology, absolute and proportional enumeration of the specialized
cells and .functional assays. The extra chromosome 21 in OS likely has a
direct or indirect effect on most immune abnormalities which are
observed in OS.
Blood Cell Populations
The microhematocrit method is used widely to yield precise
estimates of the percentage of the blood which is occupied by the
cellular component ( Oavidsohn and Nelson 1969). Abnormally high hematocrits have been reported in association with OS (Miller and
Cosgriff 1983), suggesting some hematopoietic defect. Establishing hematocrit levels will also give information regarding the physiological
condition of each individual studied.
A total WBC count provides a basis for interpreting enumeration of
subpopnlations of leukocytes. Fluctuations in WBC counts are common at
all ages, but are greatest in infants and children (Oavidsohn and Nelson
1969). Total numbers of WBC and lymphocytes have been reported to be 8
low in DS children in one study (Hann et al. 1979) while other studies
have found them to be present in normal amounts in both adults and
children (Spina et al. 1981; Dela Nuez et al. 1982).
Differentiation of granulocytes, lymphocytes, and monocytes is made
by counting cells on a stained smear. Information regarding the
relative proportions of the cell types is useful when looking for a
deficiency in the immune response, but care must be taken to interpret
these results in terms of the total WBC count. Differential l eukocyte
counts in DS have been reported to be similar to those in normal
controls (Rosner et al. 1973).
The Phagocytic System
In DS, the phagocytic sys tern appears' deficient. Although the
~•antitative n~•trophilic response to infection generally appears normal
(Levin 1979), there has been a report that the percentage of
polymorphonuclear leukocytes is low in DS adults (Spina et al. 1981).
Both monocytic and neutrophilic chemotaxis appear decreased in DS
children and young adults (Barroeta et al. 1983; Barkin et al. 1980a;
Bjorksten et al. 1980). Phagocytic and bactericidal capabilities of
neutrophils are diminished in DS (Rosner et al. 1973; Kretschmer et al.
1974; Costello and Webber 1976; Barkin et al. 1980b), however, monocyte killing seems normal (Barkin et al. 1980b).
The Antibody-mediated System
The number of circulating B lymphocytes (B cells) in DS is controversial; some workers report low levels in adults (Walford et al.
1981; Hann et al . 1979; Spina et al . 1981; Franceschi et al. 1978) while others report normal or elevated levels i n children and adults 9
(ilhittingham et al. 1977; Dela Nnez et al. 1982; Levin et al. 1975;
G•rshwin et al. 1977). Reports concerning the levels of the different inmunoglobulin (Ig) classes in DS are conflicting (Nishida et al. 1978;
O.la Nuez et al. 1982), but the most consistent findings are that IgM is d•creased, IgG increased, with IgA increased occasionally (Steihm and
F1denberg 1966; Hann et al. 1979; Kaldor and Pitt 1971). In contrast,
Mi ller and co-workers (1969) observed low IgG in DS newborns, and s1ggested that the increased IgG seen often in older subjects may be compensatory to a primary defect in IgM synthesis.
Siegel (1948b) noted that antibody response to specific vaccines is stppressed in DS patients, but more recently normal responses have been r.ported (G,riffiths and Sylvester 1967; Hawkes et al. 1978; Hawkes et a1. 1980). Also naturally occurring antibodies, specific for various m tigens , are present at normal levels in DS individt1als (Sever et al.
1}70; McKay et al. 1978). Observations of increased incidence of a'ti -thyroid anti body have been reported ( Fialkow 1970), but differences b• tween DS patients and healthy subjects are small, and the mothers and sl blings of DS individuals also exhibit anti-thyroid antibody (Fialkow et al. 1965). Therefore, the importance of these findings is uncertain.
T!e Cell-mediated System
Cell-mediated immunity for the most part is mediated by T cells.
~e characteristic of T cells is their possession of surface receptors br sheep erythrocytes (SRBC). Most studies of DS individuals, i1cluding newborns and young children, have shown a deficiency in the mmber of cells which will form rosettes with SRBC (Burgio et al. 1974;
~vin et al. 1975; Franceschi et al. 1978; Griffiths et al. 1969; 10
Gershwin et al. 1977; Dela Nuez et al. 1982; Levin et al. 1979),
Franceschi et al. (1978) reported that with the reduction in T and B
cells in young DS adults, there is an increased proportion of third
population cells ("non-T, non-B" cells; a,k .a. "null" cells) which lack
both T (SRBC receptors) and B cell (surface Ig) markers. In contrast,
one group recently reported normal numbers of rosetting cells in young
DS adults (Spina et al. 1981) while another group reported normal T cell
numbers as identified by a pan-T cell (9,6) monoclonal antibody in children (Gupta et al. 1983). When SRBC were treated with
2-amino-ethyl-isothiouronium bromide (AET) prior to rosetting with lymphocytes, levels of rosetting cells in DS adults appeared elevated
(Whittingham et al. 1977), The putative effect of such pretreatmeQt is
to enhance the size and stability of the rosettes (Bloom et al. 1976).
An interesting finding is that many of the third population cells in DS possess low-avidity receptors for SRBC and membrane receptors for autologous erythrocytes (Burgio et al. 1978). Perhaps AET affects the low-avidity receptors in such a way that the avidi t y for SRBC is increased. It has been suggested that most third population cells in DS are immature T cells differentiating along the thymus-dependent axis,
Because of this idea and the conflicting reports in DS, a comparison of rosetting methods with and without AET-pretreatment of SRBC may be worthwhile.
Assays of in vitro T cell function are especially useful when dealing with human subjects. In DS, mitogenic stimulation of lymphocytes by PHA has been studied extensively . A vast majority of studies show a decreased lymphocyte response to PHA mitogenesis in DS adults (Agarwal et al. 1970; Burgio et al. 1975; Gershwin et al. 1977; 11
Whittingham et al. 1977; Mellman et al. 1970; Franceschi et al. 1978;
Spina e t al. 1981; Walford e t al. 1981). It has been suggested that this deficient response may reflect excessive suppressor cell activity
(Thase 1982), but deficient helper cell function could also be the cause. However, decreased nnmbe rs of the suppressor /cytotoxic T ce 11 subset defined by the OKT8 monoclonal antibody have been reported in
2-12 year old DS subjects, while normal levels of the OKT4+ helper/inducer ~1bset were seen (Q1pta et al. 1983). More recently, DS individuals whose cells showed a low response to PHA were shown to have a "reversed" T4:T8 ratio (i.e. < 1.0). The low ratio appeared to primarily be due to a higher than normal proportion of OKT8+ cells in
11 the group of "low responders • DS individuals ,which responded normally to PHA had T4:T8 ratios > 1.0 (Karttunen et al. 1984). It is not known whether the abnormal proportions in the OKT8+ subset are dne to altered
011mbers of S11ppressor cells or cytotoxic cells . Helper f,tnction has also been r eported to be intact in DS adults (Whittingham et al. 1977).
Studies which have looked specifically at DS children report that
PHA-induced proliferation is normal, declining only at some t ime after the first decade of life (Dela Nuez et al. 1982; Burgio e t al. 1974;
Seger et al. 1977; Nishida e t al. 1981). There are only a few reports of the mitogenic effect of other plant lectins in DS. Whereas PHA preferentially stiw1lates T helper cells, Con A and PWM exert a more general stiw1lation on T cells. Con A is known to sti~1late both the helper and suppressor T cell subsets. The responses to Con A and PWM are reported to be reduced in DS adults (Whittingham et al. 1977;
Gershwin et al. 1977). One study found that these reductions are age-dependent (Nishida et al. 1978). Another showed tha t PWM, at 12 optimal and supra-optimal doses, preferentially stimulates T cells , while sub-optimal doses will stimulate ll cells (Franceschi et al. 1978).
The T cell re8ponse to PWM is reported to be low in DS while the B cell response appears normal.
T cell proliferation in response to autologous cells in DS adults may be quite reduced (Franceschi et al. 1981), but it has been reported that DS children 2-12 years old respond normally (Gupta et al. 1983).
Similar con~1sion exists regarding the proliferative response t o alloantigeM , with one group reporting an abnormally high response in DS
(Sasaki and Obara 1969) and another observing low response in 2 of 3 age groups (Walford et al. 1981).
The producti.on of certain l ymphokines in DS has been investigated.
In vivo production of leukocyte migration inhibition factor is reported to be low in DS infants (Levin et al. 1977). In vitro PHA-indnced l~tkocy te migration inhibition factor is depressed in DS infants and children, while it appears normal in DS adults (Levin et al. 1979). The production of both PHA-induced immune IFN (gamma) and classical vi r;,l
IFN (alpha), induced by poly I:C, is reported to be reduced in DS (Levin et al. 1979; Boyer and Fontes 1975). However, in children 4-10 years old various IFN inducers triggered higher levels of production of alpha and gamma IFN than in normal controls (Funa et al. 1984). Production of
: nterleukin-2 (IL-2) by PHA-stimulated mononuclear cells was reported to be normal in a group of 18 DS individuals ranging in age from 5 to 37 years (Karttunen et al. 1984). Also, levels of cyclic adenosine monophosphate, an immnne mediator, have been reported to be decreased in
)$ lymphocyte membranes (Levin et al. 1979).
The ability to spontaneously kill certain types of tumor cells 13
been attributed to a subset of the third pq:>ulation cells, usually
referred to as NK cells. Several studies report normal NK function in
DS (Spina et al. 1981; Matheson et al. 1981; ~i1rmi et al. 1982; Walford
1982), however all of these studies were done in adults. Speculation
that NK cells are an immature form of T cells is common.
All the observations of abnormal T cell numbers and function in DS
have led rese archers t o suspect defective thymic capacity . In fact,
morphologic derangement in the thymus and low levels of thymic hormones have been reported in DS (reviewed by Ugazio 1981).
Zinc and Immunity in Down Sy ndrome
It is known that nn tri tion has a complex influence on immune
function; specifically, the single elerrent zinc has a profonnd effect on
thymus-derived immunity ( Go od 1981). The thymus, T cells and cellular
imrn1nity in bo th humans and animals appear to be uniquely sensitive to
this sin~le trace element. Cattle with genetically caused ma1absorp tion
of zinc have a markedly hypoplastic thym1s, deficient T cell immunity
and increase d susceptibility to zinc (Bmmmerstedt et al. 1977). The human disease acrodermatitis enteropathica is also a genetically caused
zinc deficiency with associated problems including extreme susceptibility to infections. Supplementation of patients diets with
zinc will completely cure the patient of the disease (Moynahan and
Barnes 1973).
Because of the extensive thymic abnormalities observed in DS (Levin et al. 1979; Ugazio 1981) it is a possibility that a zinc deficiency may exist. Conflicting reports have appeared regarding zinc levels in the plasma of DS individuals. Two studies showed significantly decreased 14
levels of zinc (Milnnsky et al. 1970; Bjorksten et al. 1980). In one of
those stndies (Bjorksten et al. 1980) the diets of OS patients were supplemented with zinc. S11pplementation resulted in increased serum zinc levels, normalized phagocytic cell fnnction and partial restoration of certain T lymphocyte-mediated imnn1ne fnnctions. Another stndy of plasma zinc fm•nd no evidence of altered levels in OS (Neve et al.
19 83a) .
Gene-loci on Chromosome 21
It is interesting to note that the location of the gene which codes for the IFN snrface receptor in hnman cells has been determined to be on the distal end of the long arm of chromosome 21, the same segment which seems to be responsible for the DS phenotype (Epstein and Epstein 1976;
Revel et al. 1976). In cnltnres of fibroblasts from trisomi c OS individnals the antiviral activity of IFN is increased (OeClerq 1978).
It has been observed that there is increased IFN-indnced i nhibition of thymidine nptake by lymphocytes from OS adnlts , when stinn•lated by PHA,
Con A, or PWM (Epstein and Epstein 1980; Cnpples and Tan 1977; Matheson et al. 1981). However, nptake is enhanced when cells are stimnlated by tetanns toxoid (Epstein and Epstein 1980) or Staphylococcns Cowan I
(Matheson et al. 1981). Monocytes from OS patients are more sensitive to IFN inhibition of their matnration to macrophages (Epst.ein et al.
1980). IFN is also known to angment NK ac ti vi ty. Reports are somewhat conflicting as to whether cells from OS patients are more sensitive to the NK-angmenting effect of IFN. One gronp fonnd no difference between
OS and control individnals (Matheson et al. 1981) while another gronp fonnd that the angmentation of NK activity in cells from adnlts with OS 15
is dependent on the presence of monocytes (M,rmi e t al. 1982). A more
recent report states that NK cells from DS children are more sensitive
to angment~t i on by IFN (Fnna et al. 1984). Where IFN is more effective
in DS, the effects are not always proportional to the gene-dosage (a 50%
increase), a phenomenon yet to be explained .
The gene-locns coding for the enzyme snperoxide dismutase (SOD) is
also assig ned to the long arm of chromosome 21 (Tan et al. 1973).
Supe roxide is an oxygen metabolite produced by phagocytic cells for it's
bactericidal activity. The normal role of SOD is the conversion of the
superoxide radical to a less toxic chemical species so as not to render
harm t o the host tisst1es. Increased activity has been demonstrated in
several DS cell types (Neve et al. 1983b; Feaster et al. 1977). The SOD
activity in DS is usually a predictable 50% higher than in normal
cont rol::;. This increased SOD co-exists with many features of DS that
may be secondary to oxidative damage . Such damage is thought to
contribute to rapid aging, which in turn may lead to immunodeficiency
(Sinet 1982).
Immunity in Down Syndrome Children
In summary, only a few studies have reported findings specifically
about immunity in DS children. Total numbers of WBC and lymphocyte numbers have both been reported to be low, but WBC counts normally
fluctuate in this age group. Decreased phagocytic cell chemotaxis has been observed in a group of DS children. The number of circulating B cells is reported to be normal or elevated, but t here is one report of
low serum IgM in DS newborns. DS children and their siblings and mothers have increased thyroid antibody levels. Newborns and young 16 children with DS are reported to have low numbers of rosette-forming cells (RFC or T cells), however there has been a report of normal
numbers of T cells, in DS children, as detected by a pan-T monoclonal
antibody. One group reports that DS children have a low proportion of
OKT8 + cells and normal proportions of OKT4+ cells. Normal proliferative
responses to PHA and m•tologm•s cells have been reported in DS children.
Decreased production of leukocyte migration inhibition factor has been
repor ted in children with DS, but production of IFN alpha and gamma has been observed to be elevated. Finally, one report found NK cells from
DS c hildren to exhibit increased sensitivity to IFN-augmentation of NK activity. Most of these reports are solitary reports which await confi rmation or otherwise, a major p11rpose of this sbtdy. 17
MATERIALS AND METHODS
Down Syndrome Subjects and Controls
Blood samples were drawn from children, 5 years old and younger, who have trisomy-21 DS. Most of these children attend classes at the
Developmental Center for Handicapped Persons affiliated with Utah State
University. Parental consent was obtained prior to sampling according to the stipulations of the University Institutional Review Board, which gave approval to this project.
For each DS child sampled, another healthy child about the same age
(within a year), but not having DS, was sampled as a control. Sex was not matched in every case.
Collection and Separation of Blood Samples
Approximately 10-30 ml of blood was drawn with syringes containing
10 units of preservative-free heparin per milliliter of blood to be collected. Plasma was removed by centrifugation at 500 x g for 15
0 minutes and a portion of the plasma was frozen at -70 C for zinc determinations described below. The cell pellet was reconstituted to the original volume with RPMI-1640 (GIBCO, Grand Island, New York) containing 100 U/ml penicillin and 100 ug/ml streptomycin (medium).
About 5 to 7 ml of the cell suspension was then carefully layed over 3 ml of ficoll-diatrizoate (Histopaqne-1077, Sigma, Saint Louis) in 15 ml conical centrifuge tubes. Enough tubes were used to overlay the entire sam1,>le. The mononuclear cells were then separated by density gradient 18
c.entriti•gation at 800 x g for 20 minutes.
The PBMC interface was c.arefully removed using a pasteur pipet and
washed 2 time.• in medium, the first wash at 500 x g to get rid of the
ficoll and the sec.ond wash at 200 x g to remove the platelets. The PBMC
were then resuspended in rredinm for use in as many of the assays
desc.ribed below as possible.
Hematocrits, Total White Cell Counts,
and Differential Counts
Hernatocri ts, total WBC counts and differential smears were
performed on some of the whole blood samples before the centrifugation
for removing the plasma.
The microhematocrit method was used by filling two capillary tubes
per sample and centrifuging them for 5 minutes on a special centrifuge
to pack the cells. Linear measurements were made to calculate the
percentage of the total blood volume occupied by the packed cells.
For the total WBC c.onnt, the sample of whole blood was mixed well
and a small volume was diluted with acidic crystal violet solution. The
WBC were counted on a hemacytometer and calculations were made to obtain
total WBC concentrations.
Smears were made on microscope slides with a drop of the whole
blood sample. Wright's stain was used to stain the cells and a
differential count was performed to enumerate lymphocytes, monocytes,
neutrophils, eosinophils and basophils.
SRBC-rosetting With and Without AET-treatment
Cells forming rosettes with SRBC, RFC or T cells, were enumerated
• 19
using a resetting technique. Sheep blood was collected aseptically in a
syringe or tube with 10 units of heparin per milliliter of blood
collected . The sheep blood was then centrifuged at 1000 x g for 20
minutes and the huffy coat and plasma were removed. The SRBC remaining
were washed once more in phosphate buffered saline (PBS) or medium at
1000 x g for 20 minutes and then reconstituted with medium and stored at
4°C for up to 1 month. The PBS used here and in other procedures
described below contained CaC1 (0 . 10 g/1), KCl (0.20 g/1), KH Po 2 2 4 (0 . 20 g/1), MgC1 - hexahydrate (0.10 g/1), NaCl (8.00 g/1) and Na HPD - 2 2 4 heptahydrate (2.16 g/1).
Some of the SRBC were pretreated with AET prior to enumerating RFC.
A 4% (w/v) solution of AET in distilled water was first adjusted to a pH
of 9.0 with 4N NaOH. One volume of packed SRBC was incubated with 4 volnmes of the basic AET solution for 15 minutes in a 37°C water bath.
The cells were then washed 4 times with cold PBS and a 10% suspension of
AET-treated SRBC was prepared in medium and stored up to week at 4°C. 6 Human PBMC were suspended in medium at 5 x 10 /ml. Equal volumes of the PBMC suspension and SRBC (AET-treated or untreated), suspend ed at a concentration of 1% in medium containing 20% fetal bovine sentm (FBS), were mixed gently in a small tube, centrifuged 5 minutes at 200 x g and
refrigerated at least 15 mirnttes. For each 0.5 ml of the mixture, 1 or
2 d raps of 0.1% toluidine blue in PBS was added. The pellet was then carefully resuspended by rocking gently so as not to disturb the rosettes which had formed. Using a pipet tor wi th the end of the tip cut off so the orifice is not so small, a part of the suspension was put on a slide and covered with a cover slip. Resetting cells (cells a ttached to 3 or more SRBC) and non-resetting cells were counted microscopically, 20 until a total of at least 200 lymphocytes had been counted so that the percentage of the lymphocytes which are T cells could be calculated.
Resetting of lymphocytes using both AET-treated SRBC and untreated
SRBC was done concurrently. This allowed for a comparison of the two methods.
Enumeration of T Lymphocyte Subsets
Proportions of T cell subsets were determined using the complement-dependent cytotoxicity (CDC) assay. PBMC were depleted of monocytes by incubating them in a plastic culture flask for 1 hour at
37°C. Non-adherent cells (lymphocytes) were then poured off into a 7 tube and adjusted to approxima tely 1 x 10 cells/ml in medium with
10% FBS.
Commerc ially prepared monoclonal antibodies (OKT4 and OKTB, Ortho
Diagnostics, Raritan, New Jersey) had previously been diluted 1:150 and stored in aliquots at -70°C. An aliquot of each was thawed and 12.5 ul were placed in duplicate wells of a flat-bottomed 96-well microplate
(Corning). As controls, a similar quanti ty of medium alone and a pan-leukocyte monoclonal anti body (7. 29 .5, New England Nuclear, Bas ton,
Massachusetts) were also placed in separate duplicate wells. Fifty ul of lymphocytes were then added to each of the wells. The plate was incubated 1 hour at 4°C and then 50 ul of rabbit complement (Pel-Freez,
Rogers, Arkansas), which had been tested previously for it's cytotoxic capability, was added to each well. The plate was then incubated another 2 hours at room temperature.
After incubation, 50 ul of the cell suspension from one of the wells was mixed with 25 ul of a 0.4% (w/v) trypan blue solution in PBS. 21
A portion of this mixture was loaded onto a hemacytometer and dead and
viable cells were enumerated microscopically. Dead cells take up the
stain and appear blue, while viable cells exclude the stai.n. Each of
the several wells were stained and counted in this manner and the
proportions of OKT4+ and OKT8+ cells were calculated.
To test the reliability of the CDC system described above, a
commercial kit for quantifying OKT4+ and OKT8+ cells (Qt1antigen,
Bio-Rad, Richmond,CA) was obtained, and CDC and Qt1antigen were compared on a few samples. Directions which came with the Qt1antigen kit were
followed for the most part. Briefly, washed PBMC were suspended in 7 medium at approximately 1 x 10 /ml. One hundred ul of the cell suspension was added to a small test tube. A suspension l"hich had microscopic yellow latex beads coated with OKT8 monoclonal antibody and red beads coated with OKT4 antibodies was provided with the kit. Two hundred ul of the latex bead suspension was added to the tube and it was centrifuged at 150 x g for minutes, then incubated 30 minutes at 37°C.
At this point, 200 ul of a vital stain (erythrosin B) solution, provided with the kit, was added. Using a pasteur pipet, the pellet was resuspended, being careful not to use force sufficient to break up any latex bead rosettes that may have formed around OKT4+ or OKT8+ cells.
The suspension was then examined microscopically and the number of lymphocytes forming rosettes (3 or more attached latex beads) with yellow beads (OKT8+) or red beads (OKT4+) or not forming rosettes, were counted. A total of 200 cells were counted and the percentages and ratios were calculated. 22
Mitogen-stimulated Lymphocyte Proliferation
The blastogenic response of PBMC to various concentrations of 3 different mitogens was tested. The mitogens and concentrations used were as follows: PHA (GIBCO) at 1.0, 0.5 and 0.25% (by volume of commercial stock concentrate); Con A (Flow Laboratories, McLean,
Virginia) at 5.0, 2.5 and 1.25 ug/ml; PWM (Sigma) at 0.06, 0.03 and
0.015% (w/v). The mitogens were diluted in medium to twice the concentrations listed above. They were then added to triplicate wells of a 96-well flat-bottomed microplate (Corning), 0.1 ml/well. Plain medium was added to control wells.
PBMC were suspended in medium with 20% FBS at a concentration of 6 1 x 10 /ml and 0.1 ml of the cell suspension was added to each well.
The plates were incubated for 72 hours at 37°C. During the last 4 hours of incubation, the cells were pulsed with 0.4 uCi of 3 [ H]-thymidine which had a specific activity of 2 Ci/mmol (New
England Ntclear). The cells ...,re then harvested on glass fiber filter paper disks using a Skatron cell-harvester (Flow, McLean, Virginia).
The disks were placed in scintillation vials, about 2 ml of scintillation cocktail (Scintiverse E, Fisher Scientific, Fairlawn, New
Jersey) was added to each vial and the radioactivity was determined using a Packard Model 3003 Tri-Garb Scintillation Spectrometer (Packard
Instruments, Downers Grove, Illinois).
Interleukin-2 (IL-2) Production
and Assay of IL-2
When PBMC are stimulated by a variety of agents, they will produce 23
T cell growth factor, now known as IL-2. A combination of PHA (Type VS;
Sigma) and 4-phorbol 12-myristate 13-acetate (PMA; Sigma) was used in
this case. PMA was dissolved in pure ethanol and aliqnots containing ug/ml were prepared in medium and stored at -20°C until used. PBMC 7 were suspended at 1 x 10 /ml in medium containing 2% FBS and 0 . 05 mM
2"'1llercapto-ethanol (2-ME) (diluent). A solution containing 4 ug/ml PHA and 100 ng/ml PMA was prepared in dilnent and 0.5 ml was incubated with
0.5 ml of the cells in a well on a 24-well culture plate for 24 hours at
37°C. The supernatant was then harvested, centrifuged at 500 x g for
5 minutes, to remove any cells, and stored at 4°C until assay.
The IL-2-dependent cell line, HT-2 (a mtrine [BA.LB/c] cloned cell line originally prepared by Dr. James Watson) was kindly provided by the laboratory of Dr. C. Garrison Fathman of Stanford University. I HT-2 cells were maintained in culture using a mixbtre of 6 parts medium with
5% FBS, and 4 parts of medium conditioned by Con A-stimulated rat spleen 6 cells. This conditioned medium was prepared by suspending 1.5 x 10 spleen cells/ml in 100-200 ml of medium with 1% FBS, 0.05 mM 2-ME, 15mM
HEPES and 1 .0 ug/ml Con A. This suspension was incubated 48 hours at
37°C and harvested by centrifugation at 16,000 x g for 10 minutes at
4°C. It was then sterilized by filtration through a 0.22 u membrane and stored at -70°C.
The assay of IL-2 activity in supernatants was determined in a microassay based on the IL-2-dependent proliferation of HT-2 cells. The sample suspected of containing IL-2 was thawed and two-fold serial dilutions in diluent »:!re prepared in triplicate wells of a 96-well flat-bottomed microplate (Corning), 0.1 ml per well. Then 0.1 ml of 4 HT-2 cells at 4 x 10 /ml in diluent was added to each well. The plate 24
was incubated for 24 hours and during the last 4 hours, the cells ""'re 3 pulsed with 0.4 uCi of [ H]-thymidine which had a specific activity
of 2 Ci/mmol (New England N•clear). The cells ""'re harvested onto
filter paper disks as described in the proliferation assay above and
radioactivity was counted using liquid scintillation.
Eac.h time unknown samples were tested, a serial dilution of a
standard IL-2 solution was also tested. Results ""'re analyzed by probit
analysis, as described in the statistical methods below, and expressed
as half-maximal units/ml. One unit is defined as that amonnt of IL-2
ac ti vi ty which wi 11 s ti rru late a response by indicator cells which is SO%
of the maximal response.
Initially, the HT-2 cells in the IL-2 assay wonld not incorporate
the labelled thymidine and appeared dead. Finally, as described above,
2-ME was included in the medium for stimnlating IL-2 prodnction and for
the IL-2 assay. For comparison, the assay of IL-2 prodnction was done with one sample of PBMC, both with and withont 2-ME inclnded.
Assay of Natural Killer Cell Activity
and Interferon Augmentation
51 A cr-release assay was nsed to qnantify NK activity. This assay was performed rontinely the day after blood samples were drawn and
PBMC were incubated overnight (18 honrs) at room temperature in medinm with 10% an tologous plasma. With some of the samples an eqnal nnmber of
PBMC ""'re incubated with 1000 U/ml of human IFN-alpha (Sigma).
The next day, target cells, K562 cells (myeloid tnmor line), were 51 labelled with cr. Five to 10 ml of a snspension of K562 cells, which had been maintained in medinm supplemented with 10% FBS, was 25
centrifuged for 5 minutes at 500 x gin a 50 ml centrifuge tube. The 51 s1pernatant was decanted and 0.1 ml of [ cr ]-sodium chromate
rolution containing 1 mCi/ml and having a specific activity of 300 to
~00 mCi/mg (New England Nuclear) was added to the cell pellet. The
pellet was then gently dispersed and this mixture was incubated for t.our at 37°C.
During the incubation to label the target cells, the PBMC (effector cells) were washed 1 time in medium and suspended, in medium 6 s1pplemented with 20% FBS, at a concentration of 5 x 10 /ml. A
\ulume of 0.1 ml of effectors was placed in triplicate wells on a
96-well round-bottomed microplate (Corning) for a 50:1 effector to target cell ratio. Two-fold dilutions ,...re then carried out, using
~tedium with 20% FBS as diluent, and triplicate wells were prepared for effector to target cell ratios of 25:1, 12:1 and 6:1. Medium with 20%
FBS was used for background release wells and 0.5% saponin was used for maxinum release wells.
After 1 honr of incubation, the target cells were washed 3 times in a 50/50 mixture of medium and PBS and ""re resuspended at a 5 concentration of 1 x 10 /ml. One-tenth ml of the labelled target cells was then added to each well and the plate was incubated at
37°C for 4 hours.
After the incubation, 0.1 ml was carefully harvested from each well so as not to get any of the cells, .tlich had settled to the bottom of the well. These supernatants were placed in separate vials to be counted in a gamma-counter (Beckman, Irvine, California). The percent 51 of cr released dne to NK activity was calculated according to the following equation: 26
experimental release - background release % release X 100, maximum release - background release where experimental release was the mean counts per minute (cpm) from the triplicate sample wells, background release was mean cpm from the wells with medium and target cells only and maximum release was the mean cpm from the wells with saponin and targets cells only .
Results from IFN-treated cells were compared with the results from corresponding untreated cells and the extent to which the ·NK activity was augmented by the IFN was calculated.
Assay of Plasma Zinc
Zinc content of the plasma was assayed by atomic abs~rption spectrophotometry using an Atomic Absorption/Atomic Emission
Spectrophotometer 457 with a single slot burner head and a hollow cathode lamp (Instntmentation Laboratory, Wilmington, Massachusetts).
The assay used an oxidizing lean flame with an air-acetylene gas mixture and the spectrophotometer was set at a wavelength of 213.9 nm. A standard curve and the concentrations of the samples were calculated automatically by the instrument . The spectrophotometer was standardized with solutions containing 0 .1 , 0 . 2, 0.3 and 0.4 mg of zinc per liter.
They were prepared from a stock solution of 1000 mg/1 by diluting with a glycerol/distilled water mixture (5/95 by volume) to correct for viscosi ty of plasma samples according to the method of Smi th and
&ttrimovitz ( 19 79) .
Samples of plasma which had been collec t ed and frozen at -70°C as described above were then thawed and diluted 1:10 in distilled water.
After thorough mixing, the zinc content was determined with the atomic 27 absorption spectrophotometer.
Statistical Methods
Data from the assays enumerating the various subsets of cells were analyzed using a paired sample t-test (Zar 1984). An exception was the chi-square analysis (Bishop 1980) used to test for significant differences in proportions of cells obtained by the differential counts.
A 2-way factorial randomized block split plot design was used to analyze variances in the several tests which involved w1ltiple treatments within groups of age-matched subjects. These included mitogen proliferation, enumeration of SRBC-rosetting cells with and without treatment by AET and NK cell function and it's augmentation with
IFN.
The model for this analysis expressed as:
where u is the mean, P designates age-matched pairs, C represents the two conditions of DS and the control group, Tis derived from the
~1ltiple treatments in each of the experiments, CT is the interaction between conditions and treatments and E and 8 are error terms. This analysis was computed using a program called FCTCVR.
In addition, the slopes of the regression lines of NK cell activity with and without IFN treatment were compared using analysis of covariance (Snedecor and Cochran 1967).
Results from the assay for IL-2 activity were derived using probit analysis (Gillis et al. 1978). Briefly, this was done by calculating what percentage of the maximal response each individual response gave.
This produced a sigmoidal response curve which was then linearized by 28 transforming the percentage values to probit units . Tables for this transformation have been published (Finney 1971). The resulting values were plotted with probit units on the vertical axis and log of 10 reciprocal dilutions on the horizontal axis. The original percentages can also be plotted directly on probability paper. A probit regression line was then calculated and the dilution which contained 1 half-maximal unit was the anti-log of the value on the x-axis which corresponded with
SO% (5 probit units) on they-axis. From this, the concentration of
IL-2 activity in the undiluted sample was obtained. The results thus derived were then analyzed using the paired sample t-test mentioned above. 29
RESULTS
Enumeration of Various Cell Populations
in the Blood
For cells in t he blood to be effec t ive in defending the body
against invasive organisms or oncogenic transformation, they not only mtst be fnnc.tional, but the nust also be present in adequate and
~propriate numbers. For this reason various cell types were ennnerated
in this study. Table lists the results of studying erythrocyte a nd
leukocyte levels in the blood of ymng DS and control subjects.
Hematocrits of children with DS were significantly higher than those of healthy children (p < 0.05). Total WBC concentrations for the two f(roups were similar and well within the normal range. A differential count of the WBC revealed a very significant difference between the proportion of cells from DS and control m•bjects, as analyzed by chi-square analysis (Table 2). Partitioning of this chi-square (Table
3) revealed that all of the difference was due to differences in the proportions of the lymphocyte and neutrophil subsets. However, when the absolute numb ers of circulating lymphocytes and neu trophi ls were calculated by m1ltiplying the percentage obtained in the differential count by the total WBC count for each individual, it became apparent
that there was no significant difference in the numbers of neutrophils
(Table l ), In addition, when analyzed by the paired t-test, the difference in the number of lymphocytes in samples from DS and control children is only marginal (p < 0.1). 30
Table 1. Enumeration of blood cell subsets in ymng children with Down syndrome ( DS)
a Number of Subject Group matched pairs Cell population studied Down syndrome Control
Hematocrit (% of vol.) * 13 42.6 (6.o} 38.7 (4.4)
Total white bloo'6 cell count (X 10 /ml) 14 7.6 (2.6) 9.8 (4 .9)
Differential cmnt 8 ( % of total WBC) Lymphocytes *** 35.3 (12 .8) 49.6 (10.1) Neu trophils *** 53.9 (9.7) 39.5 (13. 7) Eosinophils 1.5 ( 1.6) 1.4 (1.7) Basophils 0.6 (0.7) 0.8 (1.2) Monocy tes 8.8 (6 .0) 8.8 (9.0) 6 (x I 0 /ml) + Lymphocytes 2.9 (1.8) 4.7 (1.9) Neutrophils 4.2 (1.6) 4.2 (3.9) Eosinophils 0.1 (0.1) 0.1 (0.1) Basophils 0.1 ( 0 .I) 0.1 (0.1) Monocytes 0.6 (0.4) 0.7 (0.7) c SRBC-roset ti ng cells (% of lymphocytes) 9 66.3 (15.8) 70.0 (9 .7)
aDS patients and age-matched controls,< 6 yrs old.
bMean (standard deviation).
cSRBC were treated with AET before rosetting procedure.
+ p < 0.1, by paired t-test analysis.
* p < 0.05, by paired t-test analysis.
*** p < 0.001, by chi-square analysis. 31
Table 2. Chi-square analysisa of data from differential blood cell counts in young children with Down syndrome
H : There is no difference in the proportion of cell 0 subsets in the blood from Down syndrome (DS) and age-matched control (CN) children.
Lympho- Neutr- Eosin- Baso- Mono- Total cytes ophils ophils phils cytes Obs.
Obs. 564 862 24 10 140 1600 DS Exp • 679 747 23 - 11 140 Dev. -115 115 1 -1 0
Obs. 794 632 22 12 140 1600 CN Exp. 679 747 23 11 140 Dev. 115 -115 -1 1 0
Total 1358 1494 46 22 280 3200 Obs.
2 ( Obs- Exp) chi-square = 2: Exp 74 . 7
df (U of columns - 1)(# of rows - 1) 4
p < 0.001
aSee Bishop 1980, pp. 72-78. 32
Table 3. Partitioning of chi- square analysisa of data from differential blood cell counts i n young children with Down syndrome
H : There is no difference the proportion of cells subsets 0 in the blood from Down syndrome (DS) and age-matched cont rol ( CN) children .
Lympho- Neu t r- Total Eosin- Baso- Mono - Total cytes ophils Obs . ophils phils cytes Obs .
Obs. 564 862 I426 Obs . 24 IO I40 174 DS Exp. 679 747 DS Exp . 23 II I 40 Dev. -II5 115 Dev. I -I 0
Obs . 794 632 1426 Obs , 22 I2 14 0 174 CN Exp . 679 74 7 CN Exp. 23 II 140 Dev. II5 -II5 Dev . -1 1 0
Total Total 1358 I494 2852 46 22 280 348 Obs . Obs.
chi - square 74 .4, df = 1 chi- square = 0.3, df = 2
p < O.OOI not significant
aSee Bishop 1980, pp. 72 -78 . 33
Among the lymphocytes from the DS children, a lower mean percentage of cells forming rosettes with SRBC was observed (Table 1), but the difference was not significant. The SRBC used in this study were p r etreated with AET, reputedly resulting in more stable rosette formation. In earlier stndies of RFC in DS, SRBC may or may not have been treated with AET. Since some of the SRBC receptors on T cells from
DS individuals might have a lo""'r avidity for SRBC, rosettes f ormed with non-AET-treated SRBC may be more prone to disrupted during the procedure prior to counting. This wonld result in erroneots counts and reports of lower levels of RFC. A comparison of T cell enumeration with and without AET treatment did not reveal any difference Figure 1 shows that young DS children have significantly lower T4:T8 ratios than control children (p = 0.01). Of 16 children with DS, 4 had "reversed " ratios; i.e. T4 :T8 < 1.00. The imbalance could be due to a low proportion of OKT4+ cells, a high proportion of OKT8+ cells, or both. In fact, both an increased proportion of OKT8+ cells (p < 0.02) and a decreased proportion OKT4+ cells (p < 0.05) ""'re observed in the DS group. However, when the absolute numbers of these two subsets were calculated, by multiplying the percentage of each subset by the number of circulating lymphocytes (this >~as determined from the differential count a nd the total WBC count) for each individual, it was seen that the numbers of OKT8+ cells in samples from the DS children were similar to the numbers seen in control samples (Figure 2). The paired t-test 34 Table 4 . Comparison a of two techniquesb for enumerating rosette-forming cells in young Down syndrome children Percent Rosette-forming Cells Subject Groupe Untreated AET-treated SRBC SRBC Down Sy nd r'1me 58.3 (3.1)d 64.1 (5.4) Control 69.1 (2.7) 70.2 (3.2) aNo statistically significant differences were observed between the two techniques being compared. bSheep red blood cells (SRBC) which had not been treated with AET (see methods) were used in the standard resetting technique in parallel with AET - treated SRBC. cEight age-matched individuals < 6 yrs old from each group were tested. dMean (standard error of the mean) . 35 5 • .!! 4 Q) u + C() 1- ~ 0 3 • 0 • --en • • G) u + ~ 2 ii ~ • 0 I • -0 0 • :;: I • I 0 a::: • • • • OS c Figure 1. Depressed ratio ofT cell subsets (OKT4+:0KT8+) in young children with Down syndrome (p < 0.01). The number of PBMC bearing each of the T cell mar-kers were enumerated with a complement-mediated cytolytic assay. Sixteen age-matched pair of children were studied. Each point represents the ratio derived from an individual child. Mean± s.e.m. is shown. 36 • jOKT4+l • loKTB+I 3 • • • 2 • • • • • :::: Q) • tJ • :::: • • • 0 u • • • • • OS c OS c F:gure 2. The number of circulating OKT4+ cells in young children with Down syndrome (OS) is significantly depressed (p < 0 . 05). OKT8+ cell numbers are the same as in healthy controls (C). Mean concentration ::1::: s . e .m. is indicated for 7 individuals from each group. 37 analysis revealed that DS children do, in fact, have a significantly lower number of OKT4+ cells in there peripheral blood (p ( 0.05) . After the T4:T8 ratios had been determined for several of the age-matched samples, a question arose regarding the validity of results from the COC assay. There fore, a commercial kit (Quan tigen), which is not dependent on complement cytotoxicity for enumeration of OKT4+ and OKT8+ cells, was obtained. A few samples ....,re tested using both methods in parallel for comparison. As can be seen from data in Table 5, the two methods gave similar results. Proliferative Response to Mito~ens The response of cells from the DS children to PHA was low when compared to cells from ag e""11latched controls (p ( 0.05) as seen in Figure 3. The overall response of cells from the DS children to Con A (Figure 4) was not significantly different from the control response when analysis of variance was perforrred. However, a paired t-test revealed that the response by cells from DS individuals to the highest (optimal) dose of Con A was significantly lo""r than the control cells (p ( 0.05). Figure 5 shows that the mitogenic response to suboptimal concentrations of PWM by cells from DS subjects is not significantly different from the response by cells from normal children . The variation in the mitogen- stirmtlation data is considerable, especially for PWM mitogenesis. Often results are converted to log for statistical analysis on the assumption that variances are 10 proportional to the means. It was not apparent that that assnmption was trne for these resnl ts, so no such transformation was done, and statistical analysis was carried out on the raw data. 38 Table 5. Comparisona of complement-dependent cytoxicity (CDC) and the Quantigen kit as methods for determining OKT4+ and OKT8+ cells and T4:T8 ratios Methodb Parameter CDC Quantigen OKT4+ cells (7. of lymphocytes) 33.8 (2.6)c 35.7 (6.3)d OKT8+ cells (7. of lymphocytes) 23.4 (3.5) 25.1 (4. 7) Mean T4:T8 ratio 1.47 (0.29) 1.42 (0.15) aNo statistically significant differences were observed between the two methods being compared. bMeans of 3 paired samples ± S.D. cPercent of cells lysed; detected by trypan blue exclusion. dPercent of cells rosetting with appropriate latex beads. 39 120 100 , I - -0 80 ..- ~ C) :J I - .!: 60 ~ L · ~., c .40 -:J u0 20 1.00 0.50 0.25 PHA Concentration (%) Figure 3 . Depressed proliferative 3esponse to phy t ohemagglu t ini n (PHA) , as counts per minu te of H-thymidine uptake, by lymphocytes from young child r en with Down s y ndrome (p < 0.05) . ( r:zl ) Down syndrome group; ( - ) control group . Lines extend 1 s . e .m . above t he mean of 10 individuals f rom each g roup. 40 so 40 .., I -0..... ~ 30 C) -c~ ~ '- C) a.. 20 fl) c -~ u0 10 5.0 2.5 1.25 Con A Concentration (ug/ml) 3 Fig11 re 4. Concanavalin A (Con A)-s titmllated H- thymidine up take by lymphocytes from young Down syndrome and control children. The difference between the DS group and the control grou p that occurred with the 5 .0 ug /ml Con A concen t ration was significan t (p < 0 . 05). ( ~ ) Down syndrome; ( - ) controls. Lines extend 1 s.e.m. above the means of 9 individ11als in each group . 41 10 8 .., -I 0.... ~ 6 G) :J -.E ::e '- G) Q.;, 4 II) c: -:::l u0 2 0.06 0.03 0.015 PWM Concentration(%) Figure 5. Stimula tion of lymphocyte prolife3ation by pokeweed mitogen, measured as counts per minute of H-thymidine uptake, in young children with Down syndrome . No significant differences were observed . ( ~ ) Down syndrome; (- ) controls . Lines extend 1 s .e.m. above the means of 9 subjects from each group. 42 Interleukin-2 (IL-2) Production Each time an IL-2 assay was done, a laboratory standard of rat IL-2 was also assayed as a positive control. Figure 6 illustrates the response curve and the method for probit analysis to derive the activity in half-maximal units/ml mich were present in the original sample. This method was used to derive each of the values in Table 6. The assay for IL-2 did not work until it was discovered that 2-ME was an essential component of the assay medium. When IL-2 production and assay ~re perfonred using medium without 2-ME, incorporation of 3 H-labelled thymidine by the indicator cells was never substantially greater than by the cells in negative control wells mich contained only medium with the cells. The simple inclusion of 2-ME in the medium provided culture conditions in which the m•rine cell line, HT-2, could respond to IL-2 making assay of IL-2 possible. Because of the delay caused by leaving 2-ME out of the diluent, only three DS children were tested for IL-2 production. Table 6 shows the results from the three age-matched pairs. Tha•gh the data are not statistically different, there is a trend which suggests that T cells from DS children produce IL-2 at higher levels than the matched controls. In addition, there is a hint that IL-2 production may be age-related since the 1 year old infants produced far less IL-2 than did the 4 and 5 year old children. Natural Killer Cell Activity and the Response of Natural Killer Cells to Interferon Figure 7 shows results of the NK assay comparing DS and age-matched 43 ,.- 25 ~ r""- :; .., 20 -.::> -= -I 0 15 "' -:;, -X ...... 10 ::::::E a.. ) u 5 'V ~ ~ ,.,rv ((;:)"" ~ h(o .;:y 0: ~ "" J "'V<.:>~~ RECIPROCAL DILUTION 7 ::::::E 95 ::J 90 :::::E 6 (/') x 80 1:::: < 70 ~ z :::::E so ::J 1... so !!> ----~ St::: 0 40 CD z~ 30 0 ..... 20 4§: u c: 10 ..... iI a.. s I ~'""""' 0.30.6 0 .9 1.2 1.5 1.8 2. 1 2.4 2.7 3 3.3 RECIPROCAL DILUTION (log10) <( {' ( ( (_ ' Fignre 6. Probit analysis of the sigmoidal response cnrve from assay of a laboratory stand ard solntion (snpernatant from concanavalin A-stimnlated rat splenocytes) of interlenkin-2 (IL-2). Counts per minute are converted to percentage of maximum response and are plotted against l og of reciprocal of 10 diln t ions on probability paper. The so:r. response give the dilution containing I nnit/ml of IL-2. 44 Table 6. Interleukin-2 productiona by stimulatedb lymphocytes from young children with Down syndrome (DS) Subject Groupe Age (mos.) Pair DS / Cont rol Down sy ndrome Control 8/12 17.6 1.6 55/55 48.3 22.9 3 60/60 98.2 57.4 aUnits/ml IL-2 produced in 24 h as derived by probit analysi s . 6 b1 x 10 PBMC/ml were incubated 24 h with 2 ug/~$ PHA and 50 ng / ml PMA in RPMI-1640 with 27. FBS and 5 x 10 M 2-ME. cNo statistically significant differences between DS and controls. 50 ~ ...... 0 .-2 40 '+- 0 ~ "-" Q) 30 (/) 0 Q) -Q) £k: 20 E ·-:J E 10 _ce 0 50:1 25:1 12:1 6:1 Effector to Target Cell Ratio 51 Figure 7. Baseline natural killer cell activt ty, induction of Cr-release from K562 cells by PBMC from young Down syndrome children and age-matched controls. No significant differences were observed. ( [ZJ) Down syndrome group; ( rszg) controls. Lines extend 1 s.e.m. above the mean of 17 individuals from each group. 46 controls. Although there seems to be a trend of lo""'r NK activity by 51 cells from the DS children, the small difference in mean cr-release at four effector to target cell ratios was insignificant. A significan t 51 increase in cr-release resulted from pre-treatment of cells with IFN (Table 7). However, the mean percent increase with IFN pretreatment in the DS group was not significantly different from the control group. In order to probe further, regression lines of these data were drawn. In Figure 8, it can be seen that IFN-treatment of DS cells may have increased the regression slope. An analysis of covariance was used t o compare slopes of the four regression lines to determine if this difference was significant. Q)en a comparison of all four regression slopes was made (Table 8), significant differences were apparent. However, .t!en the slope of regression for DS IFN-aiglllE!nted NK activity was compared directly with that of DS baseline NK (Table 9) or control IFN-augrented NK (Table 10) activity, no significant differences ""'re observed. Becan se of this it cannot be concluded that the regression slope was changed by IFN-treatlllE!nt of the DS cells, neither can it be concluded that DS NK cells respond any differently to IFN than control cells. Plasma Zinc Levels The concentration of elemental zinc in plasma samples from young children with DS and age-matched controls was determined. Figure 9 shows a higher mean for the DS group, but a paired sample t-test failed to demonstrate any significant difference (p < 0.1). 47 a Table 7. Interferon augmentation of natural killer activity i.n young children with Down syndrome b Subject Group E:Tc Down syndrome Controls NK activity without 50:1 49.1 (18.5)d 44 .5 (24.3) IFN pr51treatment 25:1 32 .9 (16.3) 29.7 (18.2) (% Cr-release 12: 1 21.7 (12 . 1) 19.0 (13.8) from K562 cells) 6: 1 14.0 (9.6) 8.7 ( 7. 7) NK activity withe 50:1 67.9 (18.2) 56.5 (22 .1) IFN pr51treatment 25:1 48.3 (16.9) 40.4 (19.0) (% Cr-release 12: 1 33.1 (14 .I) 27 .o (17 .4) from K562 cells) 6: I 18.2 (11.1) 16.0 (13 .I) * * * * * * * * * * * * * * * * * * * * * * * * * * Mean i. increase 50:1 47.3 (37 .6) 38.8 ( 31.7) with IFN 25:1 59.1 (40.9) 50 .1 (41 .9) pre-treatment 12:1 72.3 (81 . 0) 57.1 (61 .5) 6: I 63 .7 ( 135.) 92.1 (88 .9) aHuman leukocyte (alpha) interferon obtained from Sigma Chemical Co., Saint Louis, Missouri. bPBMC from Down syndrome patients and age-matched controls, < 6 yrs. old; 11 individuals per group; no significant differences were observed between the two groups. cEffector to target c5t1 ratio; effectors were PBMC from subjects and targets were Cr-labelled K562 cells. dMean (standard deviation). eCells were pre-incubated 18 h at 37°C with interferon (1000 units/ml). 48 ~ DOWN SYNDRO,.E lf'H-TREATED ~0 -0 UHTREAic.D -~ 25 -0 ..._.,~ Cl Ill 0 Cl 75 ""G CONTROL ~ E :J 50 E ..s::e (.) 25 0.796 1.097 1.393 1.699 1/E=T (log10) Fignre 8 . Regression lines to assay the effects of increasing effector to ta Table 8. Comparison of r egression slopes fo r baseline and in t e r fe ron- at tgmented nattt ral killer cell activity of cel l R from Down syndrome and con t rol subjects by analysis of covariance a (Reg r . Deviation f rom coeff . ) Regression 2 Line df ~d2 ~d2 Ld Slope df ss MS XX >C'j yy Within I DS / No 43 4 . 98 192 . 9 16158 38 . 7 42 8687 207 2 DS / IFN 43 4 . 98 272 .1 24313 54 . 6 42 9445 225 3 CN/No 43 4.98 195.7 19467 39 . 3 42 769 1 183 4 CN/IFN 43 4 . 98 223 . 0 23279 44 . 8 42 13294 317 5 168 39 116 233 6 Pooled 173 19 . 9 883.7 83217 I 44 . 4 171 440 15 257 7 Difference be t wee n s l opes 3 4899 1633 MS L7 F MSI:5 7 . 01; df 3, 168 p < 0 . 001 aSee Snedecor and Cochran 196 7, pp . 43 2-436 . 50 Table 9 . Compa r ison of regression slopes fo r baseli ne and in t e r feron-augmented natu ral ki l ler cell ac t ivi t y o f cells from Down syndrome subjec t s by analysis of covariancea (Regr . Devia t ion f rom coeff . ) Regression Line df 2:d2 2:d2 2: d2 Slope df ss MS XX xy yy Within I DS/No 43 4.98 192 . 9 16158 38 . 7 42 8687 207 2 DS/IFN 43 4 . 98 272 . 1 24313 54.6 9445 225 3 84 18 132 22 1 4 Pooled 86 9 . 96 465 40471 46 . 7 85 18762 22 1 5 Difference between slopes 1 630 630 F 2 . 92; df 1, 84 no t signific a nt aSee Snedecor and Cochran 1967, pp . 432 - 436 . 51 Table 10. Comparison of regression slopes for inte r feron- angmented natural killer cell activi t y of cells from Down synd rome and control Stlbjects by analysis of covariance (Regr . Deviation from coeff.) Reg ression 2 2 Line df 2:d2 2:d 2:d Slope df ss MS XX xy yy Within I DS/IFN 43 4 . 98 272 .I 24313 54.6 42 9445 225 2 CN/IFN 43 4.98 223 . 0 23279 44 . 8 42 13294 317 3 84 22739 271 4 Pooled 86 9.96 495 47592 49 . 7 85 22991 271 5 Difference between slopes I 252 252 MS LS F """"MSi:"3 0.93; df I, 84 not significan t aSee Snedecor and Cochran 1967, pp. 432-436 . 52 2.5 • 2 • ...... • ~ 1.5 • C> • 2- 1• c N • I± c I E (I) • • c a.. • 0.5 OS c Figure 9 . Zinc levels in plasma from young child ren with Down syndrome (DS) . There was no signifi cant difference between the DS group and controls. Twelve individuals per group are r ep resented with the mean plasma zinc concentratio n ± s .e.m. 53 DISCUSSION There hav~ been many studies of immunocompetence in DS, but confusion and contradictory results persist. The immune system in DS probably changes with age much the same as it does in other human beings. The unusually high rates of morbidity and mortality in very young DS children and older DS adults is evidence of that. Environmental factors may influence the immnne system of such individuals as the years go by, and the immune system itself may appear to compensate as the individual matures. It would make sense to study the DS immune syste m at a time of life when alteration by both of these factors is at a minimum. Therefore, this study of DS children 5 years old and younger with age-matched controls has been undertaken and is reported in this dissertation. A number of difficulties were encountered during this project. Only a limited number of DS patients under 6 years old live near enough for it to be practical to obtain a blood sample from them. There were problems which made it impossible to get samples from some of these children. Although most parents were willing to give consent, some did decline to subject their child to the drawing of blood. Sometimes it was impossible to schedule a convenient time to draw a sample from a certain child. A few times when aDS child was available, blood could not be obtained since the poor mu::;cle tone which is characteristic in DS can make venipunctnre difficult. Finally, samples of blood from children under 6 years old were usually only 5 to 15 ml. With these small volumes the number of WBC was limited so that only a few of the 54 several assays and tests could be performed with each sample . This study probed questions primarily about T lymphocyte immunocompetence in DS. However, the observation that the hematocrit is slightly higher in DS children is consistent with past findings (Miller and Cosgriff 1983). Neither blood pressure nor total blood volume was studied in these DS patients. Therefore, it is not certain whether these factors contribute to a relative polycythemia resulting in the observed hematocrit leve ls. The author knows of no report stating that hypertension or decreased fluid volume occ•1r generally with DS. The hyperviscosity of this apparent polycythemia may contribute to the development of congenital disease, bnt it is not clear how it may affect immune function. It has been suggested .that increased hematocrits in OS may arise from dysfnnction in the release of hematopoietic elements in the bone marrow (Miller and Cosgriff 1983) . If this is the case, then it is likely that the defect also exerts influence on the development of marrow-derived imw1ne cells as well. There is considerable confusion about whether the concentration of WBC in DS blood is abnormal. No significant abnormality in WBC count was observed in this study. Contrary to a previous report (Rosner et al. 1973), a very significant alteration of the proportions of lymphocytes and ne11trophil levels was observed. However, careful interpretation of the data showed t hat numbers of circulating neutrophils was normal, while lymphocyte nnmbers were marginally depressed. This caused the altered proportions of these two cell t ypes seen in the differential counts . The marginal depresson of lymphocytes observed in DS suggests the possibility that lymphopenia is a characteristic of DS. Such a condition would be in agreement with other 55 reports (Hann et al. 1979) and would have a detrimental effect on ima1ne mechanisms. The possibility of a low level of lymphocytes led to the inspection of l ymphocyte subsets. Forming rosettes with SRBC is a characteristic of T lymphocytes; this characteristic was used to differentiate between T and B cells. There have been several reports of depressed levels of RFC (T cells) in OS, but there are also some conflicting reports (see references in literat11re review). In this st11dy no significant difference in the proportion of RFC betwee n OS children and the controls was observed. However, if the 011mber of circulating lymphocytes was low and the proportions of rosette-forming and non-RFC normal, it may be deduced that there are low T and possibly low B cell concentrations in OS. The effect that treating SRBC with AET has on r e~11l t8, obtained when using the SRBC-rosetting technique was investigated. Although no significant differences in results obtained using AET-treated SRBC and untreated SRBC were demonstrated while testing lymphocytes from the control children, a marginal difference was seen while testing cells from the DS group . The possibility remains that in the enumeration of RFC in DS subjects, earlier investigators may have inadvertently excluded some T cells with low-avidity SRBC receptors. If a defect of T cell ~1nction exists in DS, ~1rther dissecting of the T cell population by functional characteristics may help determine this defect. Unique surface markers on T cells characterize different functional subsets and monoclonal antibodies to these surface markers aid in quantifying different T cell subsets. Helper T cells are known to express a marker defined by the monoclonal antibody OKT4 and 56 suppressor T cells express the marker defined by the OKT8 monoclonal antibody. The abnormal balance of these subsets in DS which were ob:;erved in this study is evidence for functional imbalance. Lower T4 :T8 ratios imply that the immune system in DS is generally suppressed. A lower number of circulating helper T cells (OKT4+) appears to be the major cause of this oversuppression and conld lead to the increased susceptibility to diseas e which is observed in DS. These r e sults agree wi th those reported by Karttunen et al. (1984) for DS individuals (5-37 y ears). However, the results of this study (0-5 yr) contrast those of ~1pta et al. (1983) who reported a decreased level of OKT8+ cells and a normal level of OKT4+ cells in DS children (2-12 years). The effec t of ag e on the abnormal expression of these T cell markers in DS is not known. It is difficult to determine a pattern based on the three studies d iscussed h e r e s ince the report (Gupta et al. 1983) which contrasts most with this report is the one which involved the study of the most similar age group. Different methods for detecting these cells have been employed by different investigators. Both Gupta's and Karttnnen's groups used monoclonal antibodies in conj unction with fluorescently labelled anti-mouse IgG, while in this study, antibody-mediated CDC was employed. When CDC was compared to a commercial kit which uses monoclonal antibody-coated latex beads, it gave similar results. The commercial kit is claimed to give results comparable to methods which use fluorescently-labelled anti body. Additionally, when rabbit complement was obtained for use in this assay, different lots were screened for adequate cytolytic ac ti vi ty. These measures provided confidence in the results obtained by the CDC assay. 57 The ability of both B and T lymphocytes to proliferate in vitro in response to antigen and plant lectins is thought to reflect in vivo functional capability .. Information regarding the functional status of lymphocytes from DS patients was obtained by studying the proliferative responses to three plant lectins. At suboptimal levels, PWM stimulates primarily B lymphocytes and the normal response observed in DS patients indicates that the patients had normal B cell function. The reduced proliferative response to PHA, which sti~1lates primarily helper T cells, indicates that young DS children may have defective T helper cell function. These results are clearly contradictory to reports that cells from DS children respond normally to PHA. A possible reason for this discrepancy is that this study has been restricted to studying DS 1 patients under 6 years old while other studies make generalizations about the first 10 to 30 years of life. It is possible that helper T cell function in DS is defective only for the first few years of life. This function may be normal from ages 5 to 30 years and then decline as premature aging occurs. Unlike the findings of Karttunen et al. ( 1984), the results of this study showed no correlation between T4 :T8 ratios and PHA responsiveness. This would indicate that low PHA responsiveness seen in DS patients is not just due to a smaller proportion of helper cells, but also to some defect in their functional capability. Another mitogenic plant lectin,· Con A, is known to sti~1late both helper and suppressor T cells. At optimal doses, helper T cells are preferentially sti~1lated, and suppressor T cells are stimulated at suboptimal doses. The proliferative response by PBMC from DS children to Con A reflected this differentiation. The significantly lower proliferative response of cells from DS patients to the optimal level of 58 Con A confirms the functional defect which was seen in the response to PHA. The normal response to the suboptimal levels of Con A indicate that suppressor T cell function is intact. Mitogen-stimulated T cell proliferation depends on the production of IL-2 and the expression of IL-2 receptors (Lotze and Rosenberg 1981). Since the proliferative response to PHA is defective in DS it would be worthwhile to test DS cells for their ability to produce IL-2. The results obtained in the study of IL-2 production were preliminary at best (Table 6). The large variation due to age was ·~anticipated. Given the age discrepancy of the 3 matched pairs which were tested, variation in IL-2 production within the two groups was too great to allow any differences between the groups to become _apparent. Therefore, no statistically s i gnificant difference between cells from DS children and age-matched controls was demonstrated. However, the trend suggests that OS individuals produce elevated, or at least normal, levels of IL-2. If this were the case, we could conclude that the proliferative defect seen in T lymphocytes from DS individuals is due to defective expression of IL-2 receptors or decreased receptor affinity for IL-2. Wakasugi and coworkers (1985) have recently demonstrated the existence of two forms of the IL-2 receptor, one with low affinity for IL-2 and the other with high affinity. A study testing many more DS children i1nder 6 years old could reveal the actual situation. Time constraints and the difficulties (discussed at the beginning of this section) of obtaining enough samples for testing were the main reasons why more results are not reported here. The initial difficulty in the assay for IL-2 activity was remedied by the inclusion of 2-ME in the medium. The use of 2-ME as an effective 59 replacement for monocytes in the culture media of various in vitro i~tne systems has been reported widely. The mechanism by which 2-ME replaces monocytic function is not well understood, however theories exist involving it's reducing power and interactions with lymphocyte surface moieties that mimic antigen presentation. In the system used in this study, the mouse-derived IL-2-dependent cells required the presence of 2-ME in order to respond to IL-2. This confirms the fact that monocytes play a role in T cell clonal expansion. In their recent report Wakasugi et al. ( 1985) indicate that monocytes are necessary to induce the switch from low-affinity to high-affinity IL-2 receptors on the surface of peripheral blood lymphocytes. In light of this and the findings of the current study, 2-ME may be an effective replacement for this function. The evidence of a defect of helper T cell function and increased suppressor T cell proportions suggest that thymts function may be altered in DS. As mentioned previously, thymic aberrations have been observed in DS and the thymts is acutely sensitive to zinc. DS children included in this study however, appeared to have normal levels of plasma zinc. The values of plasma zinc reported here are r 1983a). Again, technique may acc Spontaneous cytotoxicity, or NK activity seems to represent a major surveillance mechanism "*tich guards against tumor and virally-infected cells. The study of NK activity in DS is relevant in light of the increased incidence of leukemia and upper respiratory infections seen in 60 the DS population. Although the results presented here reveal no difference in the NK activity of DS and control children, it was thought that trisomic DS cells might be more sensitive to the NK-augmenting effect of IFN, since they are more sensitive to other effects of IFN. However, no difference in the response of NK cells from DS and control children to IFN was observed in this project. Funa et al. (1984) however, reported increased sensitivity to IFN in a slightly older group of DS children (4-10 years). Their approach was somewhat different that that reported here. Funa 's group used four two-fold dilutions of IFN and only one effector to target cell ratio, while the work reported here had only one IFN concentration and four effector to target cell ratios. ~Ina et al. compared the regression slope of their data from DS individuals with that from the control subjects and reported a significant difference. If there is in fact a significant difference between the DS group and controls, the testing of only one concentration of IFN should show that difference. This would be especially apparent if the IFN concentration was within the range of concentrations which ~ma et al. used and therefore fell on their regression lines. The data in the present study is from an NK assay using such a concentration of IFN, yet no significant difference was observed. When the data for the four different effector to target cell ratios were plotted and regression lines calculated, a slightly increased slope for the IFN-treated cell from the DS subjects resulted. If the increase of the slope were significant, it wonld mean that IFN had increased the synergistic efficiency of the NK cells from the DS subjects, while no such response was observed in the control NK cells. However, analysis of covariance did not verify that this slope was significantly different 61 from the others. Therefore, no evidence of hypersensitivity of NK cells to IFN was observed. Further work in DS based on the findings of this study could prove frnitful. The elevated hematocrit raises questions about hematopoietic deficiency and it's effect on cells destined to mediate im~•nity. Preliminary work should be done, however, to be sure more physiological factors such as blood pressure, total body fluid volume and blood viscosity in DS are well understood. These considerations would give gttidance to any fttrther investigation of the elevated hematocrit. Absolute numbers of Band T cells should be established with monoclonal antibody probes, and the relationship with B and T cell proportions should be elaborated. Certainly, more should be done to follow up on the suggestion that T cells from DS children .q_re hyporesponsive to IL-2 .. Such work ought t o involve a quantitative s~•dy of the expression of IL-2 receptors by DS cells and the ~·estion of low and high affi nity classes of IL-2 receptors. A probe into the effect of age on the ability of stiw•lated T cells to produce IL-2 would be important in understanding immunocompetence during early childhood. The relationship between the response to IL-2 by murine-derived IL-2-dependent cell lines and the requirement for 2-ME could also be a very interesting line of study. An animal model for DS ( trisomy-21) would be greatly helpful in elucidating the immune problems in DS. There are two possible systems currently being pursued. The Epsteins in California have developed a mouse strai n with a trisomy which bears some resemblance to tri somy-21 in humans (Epstein et al. 1985). Also, Lenny Maroun in Illinois is currently working on a rabbit model for trisomy which shows promise 62 (personal communication). These represent possible animal models which may greatly accelerate research on trisomic syndromes. The work presented in this dissertation has investigated several aspects of cell-mediated immunity in young children with DS. Evidence for a functional T helper cell defect has been found in addition to aberrant proportions of certain blood cell groups. These findings should provide direction for further investigation which will givens a clearer 11 nders tanding of the primary abnormalities of the immune sys tern in DS. 63 CONCLUSIONS 1. Yonng children (age < 6 years) with DS have significantly elevated peripheral hematoc:rits whic:h may arise from hematopoietic: dysfnnc:tion. This dysfnnc:tion may also effect differentiation of immune cells. 2 . Marginally low lymphocyte levels and normal proportions of RFC in DS children imply that there may be low numbers of T c:ells in these children. This may result in depressed immune r esponsiveness and may lead to abnormal control mechanisms, 3. AET-treatment of SRBC for the resetting tec:hniqne does stabilize rosettes . There is a possibility that early studies of DS using the resetting technique without AET may have been detecting only a subset of T c:ells with high avidity SRBC receptors. 4. With careful screening of complemen t and uniform execution, u se of the CDC assay to mark and enumerate c:ell populations c:an provide data comparable to methods not dependent on complement c:ytotoxidty. 5. Yonng DS children have lower T4 :T8 ratios whic:h result from lower numbers of c:irc:ulating OKT4+ c:ells. These may lead to suppressed immunocompe tence in' DS. 6. Cells from young DS subjects give a deficient response to PHA mitogenesis. This is evidence that T helper c:ell clones in DS individuals may be unable to expand at a rate sufficient for immune protection. The low response of DS c:ells to an optimal dose of Con A tends to confirm a functional helper T c:ell impairment. 64 7. Low T4:T8 ratios and low PHA responsiveness apparently do not correlate in young DS children. This supports the theory that low response to the lectin is a functional defect rather than resulting from low numbers of T helper cells . 8. Preliminary evidence indicates that IL-2 production by T lymphocytes may be age dependent. Also suggested is the possibility that DS cells produce more IL-2 than controls. Such an abnormality would indicate that the defective proliferative response is caused by inadequate expression and/or function of IL-2 receptors on the cell surface, possibly a class of low affinity receptors. 9. Zinc was present in plasma samples from DS children at normal levels; therefore, no evidence was found that a restricted supply of zinc is the CaliSe of thymic abnormalities in DS. 10. NK function in young children with DS is normal; therefore, no evidence was seen that faulty NK activity results in the increased rates of infection and leukemia which are observed in DS. 11. NK cells from DS children are not more sensitive to the augmenting effect of IFN-alpha than cells from other children. 12. Mouse-derived IL-2-dependent cytotoxic T cell lines require 2-mercapto-ethanol for optimal response to IL-2. 65 LITERATURE CITED Aga rwal, S. S., Blumberg, B.S., Gerstley, B. J. s., London, w. T., Sutnick, A. I., and Loeb., L. A. 1970. DNA polymerase activity as an index of lymphocyte stimulation: Studies in Down's syndrome . J. Clin . Invest. 49:161-169. Barkin, R. M., Weston, W. L., Humbert, J . R., and Maire, F. 1980a. Phagocytic function in Down syndrome: I. Cl1emotaxis. J. Ment. Defic. Res . 24:243-249. Barkin, R. M., Weston, W. L., Humbert, J. R., and Sunada, K. 1980b. Phagocytic function in fuwn syndrome: II. Bactericidal activity and phagocytosis. J. Ment. Defic . Res. 24:251-256. Ba.rroeta, 0 ., Ntngary, L., Lopez- Osuma, M., Arrrendares, S., Salamanca, F ., and Kretschmer, R. R. 1983. Defective monocyte chemotaxis in children with Down's syndrome. Pediatr. Res . 17:292-295. Bishop, 0. N. 1980. Statistics fo r Biology. 3rd ed., pp. 72-78. Longman, London. Bjorksten, B., Back, 0., Gustavsen, K. H., Hullmans, G., Hagglof, B., and Tarnvik, A. 1980. Zinc and immme function in Down ' s synrlrome. Acta Paediatr. Scand. 69:183 - 187 . Bloom, B. R., Cohn, Z. A., David, J., Greaves, M. F., Jondal, M., Kunkel, H. G., Nnssenzweig, V., Remold, H. G., Schlossman, S. F., Wigzell, H. and Winchester, R . J. 1976. Evaluation of in vitro methods for characterization of lymphocytes and macrophages . Pages 3- 26 in B. R. Bloom and J. R . David, eds. 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Prentice- Hall, Inc ., Englewood Cliffs , New Jersey . 72 VITA Roger Lee Noble Candidate for the Degree of Doctor of Philosophy Dissertation: Cellular Imm1nity in Children with Down's Syndrome (Trisomy-21) Major Field: Imm1nology Per sonal Data: Born 8 May 1954, Brigham City, Utah, son of Myrvin E. and Iris M. Noble; married Nan Woodward June 5, 1981; children- Sam1el Woodward and Brett David. Education: Doctor of Philosophy -Immunology, Utah State University, 1985 . Master of Sci ence Virology, Utah State University, 19~3 . Bachelor of Science - Microbiology, Brigham Young University, 1978 . Professional Experience: Research Assistant - Utah State Uni ve rsi ty , Logan, UT, '83-'85 . Teaching A.c;sistant - Utah State Iniversity, Logan, UT, ' 83- '85 . Research Assist ant - Utah State Uni ve rsi ty , Logan, UT , ' 78- '83 . Teachi n ~ Assistant - Utah State University, Logan, UT, '78- '81 . Lah Technician Colorado Serum Company, Denve r , co, May - Aug ' 78. Professional Societies : American Socie ty for Microbiology Intermountain Branch - Alll!rican Society for Microbiology Utah Chapter - American Association for Laboratory Animal Science New York Academy of Sciences Honors: Second Place - Student paper competition, Intermo11ntain Branch, American Society for Hicr obiology, 1980 . 73 ~1blications and Papecs Pcesented: Noble, R.L., ~acnett, B. B., Spendlove , R. S., and Sidwell , R. W. 1979. Mucine cotavirus infection: Chacactecization of the disease and influence of malnutcition . Abstcact Ill in Intecmountain Bcanch, American Society foe Miccobiology, Pcogcam and Abstcacts , vol. 27. Spcing Meeting , Univecsity of U~ Salt Lake City, Utah (Apdl 1979). Noble, R. L., Sidwell, R. W., Mahoney, A. w., Bacnett, B. B., and Spendlove, R. S. 1980 . Chacactecization of the w•cine rotavin1s infection and effects of malniitrition on sensitivity to the disease . Abstcact 1110 in Intecmountain Bcanch, Amecican Society foe Miccobiology, Pcogr;m and Abstcacts , vol. 28. Spring Meeting, Bdgham Young Univecsity, Provo, Utah (Macch 1980). Noble , R. L., Sidwell, R. W., Mahoney, A. W., Barnett, B.B., and Spendlove, R.S . 1980. Rotavin•s infection in mice: Characterization of the disease and the influence of malnutrition. Fed. Pcoc . 39:889. (Abstc. /13277) . Sidwell, R. w., Noble, R. L., Morrey, J.D., Barnett, B. B., Spendlove, R. S., and Mahoney, A. W. 1981. Influence of malnutrition and alterations in dietary iron and protein on rotaviral disease in infant mice. Presented at: Intern~tion~l Symposium foe Medical Vi co logy, Anaheim, California (October 198 1). Sidwell, R. W., Noble, R. L., Marcey, J. D., Barnett, B. B., Spendlove, R. S., and Mahoney, A. W. 1982. Influence of dietary iron, protein and nutrient dilution on nntrine rotaviral disease . Page 266 in Amecican Society for Miccobiology, Abstracts • Annual Meeting, Atlanta, Geocgia (Macch 1982). (Abstr. IITlOO) . Moccey, J . D., Sidwell, R. W., Noble , R. L., Bacnett, B. B., and Mahoney, A. W. 19 82 . Effects of folic acid malnutcition on rotaviral infection in mice. Abstract 1/3 in Intermountain Bcanch, Amecican Society foe Miccobiology,:Program and Abstcacts , vol. 31. Fall Meeting, Univecsity of Utah, Salt Lake City, Utah (October 1982). Noble, R. L., Sidwell, R. W., Mahoney, A. W., Bacnett, B. B., and Spendlove, R. S. 1983. Inf~•ence of malm•tri tion and alterations in dietary protein on ®trine rotaviral disease. Proc. Soc . Exp . Biol. Med. 173:417-426. Noble, R. L. 1983. Murine cotavin•s infection: Characterization of the disease and influence of host malnutrition and dietary protein. M.S. Thesis. Utah State University . 95 pp. 74 Morrey, J. D., Sidwell, R. W., Noble, R. L., Barnett, B. B., and Mahoney, A. W. 1984. Effects of folic acid malm•trition on rotaviral infection in mice. Proc. Soc. Exp . Biol. Med . 176:77- 83. Noble, R. L., Warren, R. W., and Thain, W. S. 1984. The ontogeny of human natural killer ac tivi ty . Presented at: Seventh Annual Rocky Moun t ain Immunology Meeting, Alta, Utah (August 1984) . Noble, R. L., Warren, R. P . , and Thain, w.s. 1984. Human natural killer (NK) activity: Ontogeny and augmentation by interferon. Abstract /116 in Intermountain Branch, American Society for Microbiology,-program and Abstracts , vol. 33. Fall Meeting, Brigham Young University , Provo, Utah (November 84) . Noble, R. L., Warren, R. P., and Thain, W. S. 1985. Defective mitogenic response to phy tohemagglu ti ni n and reduced T4: T8 ratios in young children with Down's Syndrome (Trisomy-21). Page 83 in American Society for Microbiology, Abstracts • Annual Meeting,~as Vegas, Nevada (March 1985). (Abstr. II E52). Noble, R.L. and Warren, R.P. 1985. Cellular immune system in Down syndrome. Presented ati Research and Practice in Down Syndrome - First Anm•al Conference, Logan, Utah (~•ne 1985). Noble, R.L. and Warren, R.P. 1985. Age - rel~ted nevelopment of human natural killer activity. New England J Med (Sep 5 ' 85). (Letter). Noble, R. L. 1985. Cellular immunity in young children with Down syndrome (Trisomy-21). Ph . D. Dissertation . Utah State University. 83 pp.