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Mediators Inflamm. 2005 December 14; 2005(6): 317–330. doi: 10.1155/MI.2005.317. | PMCID: PMC1323332 |
Microarray Profiling of Lymphocytes in Internal Diseases
With an Altered Immune Response: Potential and
Methodology Anatoliy Gladkevich,* S. Adriaan Nelemans, Henk F. Kauffman, and Jakob Korf Departments of Psychiatry, Molecular Pharmacology, and Allergology, University Medical Center of Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The Netherlands Received April 25, 2005; Accepted August 1, 2005. This is an open access article distributed under the Creative
Commons Attribution License which permits unrestricted use,
distribution, and reproduction in any medium, provided the
original work is properly cited. Recently it has become possible to investigate
expression of all human genes with microarray technique. The
authors provide arguments to consider peripheral white blood cells
and in particular lymphocytes as a model for the investigation of
pathophysiology of asthma, RA, and SLE diseases in which
inflammation is a major component. Lymphocytes are an alternative
to tissue biopsies that are most often difficult to collect
systematically. Lymphocytes express more than 75% of the human
genome, and, being an important part of the immune system, they
play a central role in the pathogenesis of asthma, RA, and SLE.
Here we review alterations of gene expression in lymphocytes and
methodological aspects of the microarray technique in these
diseases. Lymphocytic genes may become activated because of a
general nonspecific versus disease-specific mechanism.
The authors suppose that in these diseases microarray profiles of
gene expression in lymphocytes can be disease specific, rather
than inflammation specific. Some potentials and pitfalls of the
array technologies are discussed. Optimal clinical designs aimed
to identify disease-specific genes are proposed. Lymphocytes can
be explored for research, diagnostic, and possible treatment
purposes in these diseases, but their precise
value should be clarified in future investigation. During the last 5 years, microarrays have been developed for
large-scale clinical research and routine to
identify genes involved in disease states. At present, microarrays
comprising the whole human genome (all 30 000 genes) have become
commercially available and their potential to
identify abnormal gene activity in disease is now well recognized.
The microarray approach is particularly suited to identify the
activity of genes that are differently expressed during a disease
state and that may become normalized following recovery of the
patient. So, perhaps more than with the
conventional gene-identification approach, the success of the
microarrays technique depends heavily on proper clinical
designs. The most promising gene expression
profiles have been derived from cancer research [ 1, 2, 3, 4]
using tumor tissue and peripheral blood mononuclear cells
(PBMCs). In internal diseases such as
pulmonary diseases [ 5], including asthma [ 6], autoimmune
disorders, for example, rheumatoid arthritis (RA) [ 7],
systemic lupus erythematosus (SLE) [ 8], cardiovascular
diseases [ 9], psychiatric
disorders [ 10, 11], and others
as nicely reviewed [ 12] have used mainly conventional
mRNA-assay techniques. PBMCs—and in particular lymphocytes—are particularly
convenient for medical research and diagnostic applications,
because they can easily and repeatedly be collected in sufficient
quantities in the course of the disease. The
involvement of T cells and B cells in the pathogenesis of asthma
is well recognized; particularly that of Th2 cells both in atopic
allergic asthma and nonatopic and occupational asthma
[ 6, 13]. In SLE immune abnormalities in a wide
variety of cell populations to include B and T
lymphocytes, monocytes, and natural killer (NK) cells have
been noticed [ 8]. Defects of phenotypic and
functional T cells were found in RA, including abnormal clonal
expansions and suppressed proliferative responses, which suggest a
defect in T-cell differentiation [ 14].
Excessive Th1-type cytokines have been associated with tissue
destruction as found in autoimmune disease as RA, whereas
overabundant Th2-type cytokines have been implicated in
atopy and allergic asthma. Altered function of lymphocytes in
diseases is a result of abnormal expression of genes for numerous
cytokines, receptor components, signal transduction pathways, and
modulators of transcription and translation. In addition,
polymorphism of the genes affects their functional properties.
Despite intensive studies, the pathophysiological and pathogenetic
mechanisms behind these diseases are still poorly
understood. PBMCs comprise a very heterogeneous population and the question
may arise whether meaningful information on gene expression on
particular processes can indeed be deduced from these cells. Could
it be that—for instance—in the diseased state the relative
composition of the population just changes without alterations in
the gene-expression of the individual cell per se? In
that case the changes seen may merely reflect changes in
population of cells. But even if this is the case, it should be
realized that such changes do not occur without changes in
intracellular transduction and gene expression. Activation of
sympathetic associated beta-adrenergic receptors causes
the
release of lymphocytes from the spleen. Changes in cortisol
suppress proliferation of certain subpopulations of lymphocytes.
Anyway, such receptor-mediated effects are concomitant with
altered gene expression, which is considered indicative of the
disease state. Interest of gene expression of PBMCs concerns with
identifying both pathogenetic and pathophysiological processes. Pathogenetic
processes are primarily associated with the cause of a disease, so
microarrays may lead to the identification of abnormal
genes and gene activities that may not be only limited to
PBMCs, but may occur in cells of pathological tissue as well,
or—alternatively—that the immune reaction by itself is
abnormal. In contrast, pathophysiological changes in lymphocytic
gene expression are considered as an essentially normal
reaction of the
immune system to an abnormal, that is, pathological, stimulus. So,
at least theoretically, pathophysiological gene profiles may be
shared in a variety of diseases, whereas pathogenetic gene
expression is expected to be disease specific. To discuss this
issue in more detail, we will compare lymphocytic gene expression
profiles in asthma, SLE, and RA. Because of presumably different
pathogenesis of these diseases, common gene-expression profiles
point to a general response of the immune system, whereas
differential profiles are likely to be related to disease-specific
processes. The latter is important for diagnostic applications, for research
purposes less stringent criteria may also be, and a
variety of strategies have been proposed,
so that the focus may be on a limited number or profiles
of genes
related to a particular gene. Accordingly, insight in gene
activities in the diseased state is obtained, but the chance is
relatively small to identify novel pathogenetic or diagnostic
genes. Often, the so-called supervised or unsupervised analyses
are applied. In the supervised approach all the expressed
genes are correlated with that of the expression of a known
aberrant gene, so clusters of genes involved in a particular
disease are identified. In the unsupervised approach
clusters of gene expression the structure (clustering) of genes is
determined, without a priori assumptions about gene expression.
Both strategies have recently been applied in acute myeloid
leukemia [ 3, 4]. In this review we consider the potential of the microarray gene
expression in lymphocytes as a proper and convenient approach to
investigate in detail pathophysiological processes at the
molecular level. We review the molecular mechanisms and
gene-expression patterns involved in asthma, SLE, and RA, taken
the lymphocyte as pivot. Molecular mechanisms Allergic asthma is a chronic pulmonary disease associated with
bronchoconstriction and inflammation, in which Th2 cells play a
central role. It is well known that allergen-specific IgE
synthesis is T-cell dependent on cognate activation of B
lymphocytes and Th2-derived cytokines, such as IL (interleukin)-4 and
IL-13 [ 15]. T lymphocytes of the T-helper-2 subtype,
producing IL-4, IL-5 and IL-13, have been shown to be prominent in
airways of the asthmatic patients [ 13, 16]. These cytokines
play pivotal roles in asthmatic disorder, since IL-4 induces IgE
production by B lymphocytes and IL-5 is the main factor regulating
eosinophilia. Elevated percentages of CD4 T cells expressing mRNA
encoding IL-4 and IL-5, and CD8 T lymphocytes expressing IL-5,
were found in asthmatics as compared with the controls [ 17].
IL-13 is responsible for inducing most of the phenotypic features
of allergic asthma (ie, airway responsiveness, mucus metaplasia,
and eosinophilic inflammation). The cellular targets of IL-13
responsible for each of the phenotypic features of asthma and the
specific genes whose expression is regulated in these target cells
remain, however, unknown. Since the main mode of action of IL-13
is regulating gene expression of airway tissue cells, these
questions seem ideally suited to expression array experiments
[ 18]. Moreover, it has recently been hypothesized that IL-13
may participate in the pathogenesis of asthma by activating a set
of “proasthmatic” genes in airway smooth muscle cells [ 19].
These investigators further confirm the hypothesis that gene
modulation by IL-13 in airway smooth cells is essential in the
development of allergic asthma [ 19]. Both IL-13 and IL-4 are
capable of inducing isotype class switching of B cell to produce
IgE after allergen exposure [ 20]. Many more cytokines with potential relevance to asthma have been
described. So, IL-25 acts in Th2 differentiation;
IL-9, IL-11, IL-13, IL-16, and IL-17 have been linked to asthma;
and IL-12, IL-18, IL-23, IL-27 are involved in Th1 development and
IFN- γ production, which might be deficient in patients with
asthma [ 13]. In addition, IL-12 and IL-18 have
the potential to reduce airway inflammation to inhaled challenge
after Th2 sensitization [ 21]. Data of Kodama et al point to a
role for IL-18 in allergic inflammation in which IL-18 limits the
development of the local inflammatory response to
antigen [ 22]. It has been reported that atopic asthma
is associated with a downregulation of IL-12 mRNA and also that
IL-12 suppresses the expression of Th2 cytokines and their
associated responses, including eosinophilia, serum IgE, and
mucosal mastocytosis [ 21]. It has been shown that IL-3 and
GM-CSF also may influence the inflammatory process in asthma
through their regulatory role on eosinophil survival,
differentiation, and effector function [ 23]. Furthermore
Ferreira showed that after allergic challenge in atopic patients
IL-4, IL-5, and IL-13, as well as the pro-inflammatory cytokines
GM-CSF, TNF- α, and IL-6 are consistently increased when
compared with the respective control value
[ 24]. IL-10 modulates IgE production and
induces apoptosis of eosinophils [ 25]. IL-8, TNF- α,
and leukotriene B4 can be used as markers of neutrophilic
inflammation and to evaluate the response to inhaled steroid
therapy in asthma patients [ 26]. The transcription factors T-bet and GATA3 may affect airway
immunopathology in asthma [ 27]. T-bet induces an IFN- γ
producing Th1 phenotype, and also represses IL-4 and IL-5
production from differentiated Th2 cells [ 28].
Finotto et al [ 29] provided the evidence for decreased number
of CD4+ T cells expressing T-bet in the airway of human asthma
patients relative to control subjects. Moreover,
they showed deletion of the T-bet gene in mice resulted in airway
eosinophilia, Th2 cytokine production, airway hyperresponsiveness,
and changes of airway remodeling in the absence of allergen
sensitization and inhaled allergen challenge. GATA3
could repress IFN- γ production, induces IL-4 and IL-5
[ 30], and is an important controller of the IL-5 gene locus
[ 31]. T cells from asthmatic patients
expressed 5 times the level of GATA3; so increased expression of
GATA3 may underlie augmented Th2-like cytokines in this
disease. The transcription factors c-Maf, NIP45,
and JunB that increase IL-4 activation appear to be expressed
solely in Th2-cells and not Th1 cells [ 32].
Gene deletion or overexpression of these factors correspondingly
affects IL-4 production [ 32]. Other
transcription factors might be crucially involved in gene
expression of IL-5 such as NFAT, AP-1, IL-4, IL-13, and the signal
transducer and activator of transcription (STAT6)
[ 33, 34]. Various G-protein-coupled receptors are expressed on lymphocytes.
Muscarinic receptor subtypes (M1–M5) are involved in the control
of airway function. Dysfunction of these receptors contributes to
the development of airway hyperresponsiveness and bronchomotor
responses associated with asthma [ 35]. Human lymphocytes
express M2–M5 receptors and corresponding mRNA [ 36,
37].
Ricci et al [ 38] reported that lymphocytic M2 and lesser
amount of M5 receptor subtypes are increased in bronchial
asthma and that these changes are related to bronchial
hyperresponsiveness. They suggest that peripheral blood
cholinergic receptors reflect the status of cholinergic
dysfunction and involvement of lymphocyte cholinergic
system in allergic inflammation. A reduced β-adrenergic responsiveness plays an important
role in the increased airway reactivity of asthmatic patients.
This hypothesis has been supported showing a reduced β-adrenergic responsiveness in lymphocytes of asthmatic patients,
predominantly during the occurrence of active and severe symptoms
[ 39]. The results of Motojima et al
[ 40] suggest that
beta-blockade and bronchial hypersensitivity in asthmatic patients
may in part be due to a decreased number of β-adrenoreceptors. An increasing body of evidence shows that nerve growth factor
(NGF) exerts biological activity not only on the central and
peripheral nervous system but also on the immune system, thereby
influencing allergic diseases and asthma. NGF is produced by cells
of the nervous system, and also by T and B lymphocytes, which
display functional NGF receptors [ 41, 42]. Bonini et al
[ 43] reported that NGF circulating levels of NGF are
increased in patients with allergic diseases and asthma. Moreover,
NGF increases airway hyperreactivity to histamine in an animal
model of asthma, while anti-NGF treatment reduces airway
hyperreactivity in sensitized mouse induced by topical challenge
of ovalbumin. Gene expression There are a number of candidate genes thought to play a role in
the development of asthma. The region of chromosome 5q31–33
contains several genes that modulate atopic responses, including
IL-4, IL-5, IL-13, and GM-CSF, as well as the GR and β-AR.
Polymorphism identified in the IL-13 gene has convincingly been
associated with a variation in IgE levels and with various
features of the asthmatic phenotype [ 44, 45, 46]. The IL-4 gene
is of a great interest because it causes B-cell isotype switching
from IgM to IgE and stimulates IgE production in allergic
sensitization. It has been shown that polymorphism of the IL-4
receptor gene is associated with atopy and asthma [ 47].
Moreover, gene-gene interaction between IL-4 receptor and IL-13
was associated with asthma [ 20]. The gene of the inflammatory
marker TNF- α has also been tested as candidate gene leading
to asthma [ 48]. The recent investigation of Karjalainen et al
[ 25] showed that also IL-10 gene polymorphism is associated
with eosinophil count and circulating IgE in asthma. The polymorphism of the β2-AR promotor at positions 16
(arginine to glycine) and 27 (glutamine to glutamic acid) is known
to be functionally relevant and has been associated with more
severe forms of asthma, nocturnal asthma, and decreased airway
responsiveness in asthmatic subjects [ 49]. It has been shown that corticosteroid-resistant bronchial asthma
is associated with increased c-fos expression in T lymphocytes
[ 50]. Glucocorticoids (GCs) are involved in the regulation of
numerous physiological processes and, as drugs, represent the
cornerstone of anti-inflammatory treatment in asthma. Their
anti-inflammatory effects are mediated by the GR- α which
represses expression of various genes encoding
inflammatory mediators [ 51]. Alternative splicing of
the human GR gene produces a splice variant, GR- β, termed
the silent receptor. Increased expression of GR- β is found
in GC insensitive patients with asthma [ 52]. It seems that
overexpression of GR- β may play a role in GC-resistant
asthmatics, whereas in GCs-dependent asthmatics, a predominant
GR- α expression was found. Suppression of the expression
IL-5 and GM-CSF genes by GCs has been shown in asthmatics
recovering from acute exacerbation of disease
[ 23]. CTLA-4 is a second costimulatory molecule that is expressed only
on activated T cells. It is considered to be important in the
development of many of the immunological and physiological
features of asthma [ 53]. Polymorphisms of the CTLA-4 gene
have effects on immune response in asthma and may serve as a
clinically useful marker of severe asthma [ 53]. Microarray technology offers the new opportunity to gain insight
into global gene-expression profiles in asthma, allowing
the identification of novel asthma-associated and inflammatory
genes. Differentially expressed genes in a monkey model of allergic
asthma showed that of the approximately 40 000 cDNAs represented
on the microarray, expression levels of 169 changed by more than
2.5-fold in at least one of the pairwise probe comparisons; these
cDNA encoded 149 genes, of which 52 were novel and 97 were known
genes for which a role in asthma pathogenesis had been implicated
before, such as chemokines and other inflammation-associated
genes, matrix proteins, and matrix metalloproteases, involved in
airway remodeling [ 54]. Gene expression in a mice model of
asthma revealed among the hundreds of differentially expressed
genes the unexpected observation of the increased expression of
arginase [ 55]. Gene-expression profiling airway inflammation in mice showed that
of the 1176 genes on the array, the expression patterns of 280
genes were consistently altered. Of these genes, the steady-state
levels of 93 genes were upregulated and 29 were downregulated
[ 56]. The effect of inhaled corticosteroid therapy on gene expression
was followed using bronchial mucosa biopsies from healthy controls
and subjects with allergic asthma, it appeared that corticoid
therapy normalizes partly or completely the expression of 26 of
the 79 genes of known function identified as differentially
expressed in asthmatics [ 57]. To conclude, there are several abnormalities of function and
metabolism of lymphocytes in which an altered gene expression
plays a main role. In particular, differently expressed genes
involved in inflammation remodeling and epithelium activation,
genes encoding cytokines, transcription factors, costimulatory
molecules, lymphocytic receptors, and other genes with unknown
functions were detected. But, till present, there are no
reports of microarray gene expression in human lymphocytes
in asthma. Molecular mechanisms RA is a chronic, autoimmune inflammatory disease of the synovium
with progressive destruction of affected joints. The Th1 cells are
thought to contribute to the inflammation by inducing high levels
of the proinflammatory cytokines TNF α, IL-1, IL-6, and
IL-17 in the synovium [ 58], which leads to cartilage
destruction and bone erosions [ 59]. IL-6 prompts B cells to
differentiate and mature into antibody-secreting cells. Increased
levels of IL-6 correlate with increased levels of
rheumatoid factor. Moreover, IL-6 enhances bone resorption and may
play a role in the periarticular osteoporosis characteristic of
early RA [ 60]. Also it stimulates the production of
C-reactive protein in RA, nonspecific indicator of disease
activity [ 60]. IL-4 mRNA is almost absent in RA synovium and
IL-4-producing Th2 clones can rarely be detected [ 61]. IL-2
is thought to play an important role in the pathogenesis of RA.
Serum IL-2 levels and sIL-2R were elevated in patients with RA as
compared to controls [ 62, 63]. TNF- α and IL-8 mediate ongoing destruction of cartilage,
subchondral bone, and other joint-related tissues [ 64].
Cytokine IL-18 has been cloned that exhibits powerful
Th1-promoting activities in synergy with IL-12. IL-18 induces
production by Th1 clones [ 65]. IL-18 receptor expression was
detected on synovial proliferation, upregulates IL-2R expression,
and promotes IFN- γ, TNF- α, and GM-CSF
lymphocytes and macrophages [ 66]. Small but physiologically
relevant amounts of IFN- γ and the Th1 cytokine IL-17 are
expressed in RA and could contribute to immune responses,
fibroblast activation, and bone destruction [ 67]. B cells have been shown to participate in chronic rheumatoid
synovitis. They undergo antigen-dependent clonal expansion,
affinity maturation, and differentiation into plasma cells, and
produce rheumatoid factor, a well-recognized prognostic factor
for aggressive RA [ 68]. Activation of c-fos may be involved in cartilage metabolism and
might play a crucial role in the pathogenesis of arthritic
destruction [ 69]. TNF- α and IL-6 augmented c-fos gene
expression of rheumatoid synovial cells, but transactivation of
c-fos gene became resistant against cytokine stimulation under
prolonged expression of c-fos gene [ 70]. GCs are the most powerful anti-inflammatory drugs used in the
treatment of RA. In particular, most immunosuppressive and
anti-inflammatory effects are exerted by an interaction of GRs
with AP-1 and NF-kappaB [ 71]. GCs inhibit also IL-1 and
TNF- α forming a cytokine-HPA axis feedback circuit
[ 71]. The GR number in lymphocytes might be helpful to
predict which patients with RA will response to low- or
medium-dose prednisone and therefore do not or will not require
higher doses [ 72]. An increased nitric oxide (NO) level as a result of activity of
the enzyme NO synthase has been shown in the serum and in synovial
macrophages of patients with RA. NO causes chondrocytes apoptosis
and plays a role in various other inflammatory and destructive
processes [ 73, 74]. An inducible form of this enzyme is present in macro- phages,
polymorphonuclear leukocytes, and NK cells. In murine models,
blockade of NO production prevents and treats autoimmune disease
[ 75]. Several studies have demonstrated a significant rise and fall in
the expression levels of μ- and κ-opioid receptors,
respectively, in rat with polyarthritis [ 76, 77]. In RA
patients, the κ-opioid receptor mRNA was expressed on T
and B cells and NK cells [ 78]. They also reported that the
levels of expression of κ-opioid receptor mRNA in
lymphocytes were decreased in RA patients in comparison with
healthy volunteers; and it was significantly related to the
inflammatory activity or chronic pain in the RA patients. Gene expression In RA, inflammation of the joint is caused by the gene products of
lymphocytes and other cell types from the circulating blood
presenting in the synovium and cartilage tissues. To get insights
into pathophysiological pathways, Justen et al [ 79] used the
suppression subtractive hybridization method to identify
differentially expressed genes in synovial tissue from RA
patients. DNA sequencing identified 12 gene products including
cytoskeletal γ-actin, fibronectin, superficial zone
protein, IFN- γ inducible genes such as a novel
thiol reductase,
and two genes of unknown function (HSIFNIN4, RING3).
Compared to osteoarthritis patients, 9 of the 12 genes were
overexpressed. Using microarray technology in human RA monocytes, chondrocytes,
and synoviocytes, Heller et al [ 7] showed that prominent
upregulated genes are IL-6, the MMPs Strom-1, Col-1, Ge1A, HME,
and (in certain samples) PUMP, TIMPs, and the adhesion
molecule VCAM. With the 1046-element array of randomly selected
cDNAs from peripheral blood library probes, RA samples showed
hybridizations to large number of genes. Of these three genes were
upregulated and they are TIMP-1, apoferritin light chain, and
manganese superoxide dismutase (MnSOD), while others were
differentially expressed. In the rheumatoid nodule there was a prominent expression
of IL-1 β, and TNF- α
together with IL-12, IL-18, IL-15, and IL-10 represents a
cytokine profile similar to that of the synovial lesion of RA,
which is generally accepted as being due to Th1-mediated
inflammation [ 80]. A recent study of the rheumatoid synovium using cDNA microarray
has demonstrated that a total of 121 genes were significantly
higher expressed in the RA-I tissues, whereas 39 genes were
overexpressed in the RA-II tissues [ 81]. In this study, an
attempt was made to subclassify RA patients based on the global
expression of genes in affected synovial tissue. The RA-II group
showed expression of genes suggestive for fibroblast
dedifferentiation. Within the RA-I group, two groups were
distinguished; the RA-Ia group showed predominantly immune-related
gene activity, while the RA-Ib group showed an additional higher
activity of genes indicative of the classical pathway of
complement
activation. The differences in expression profiles provide
opportunities to stratify patients based on molecular criteria
that may require different treatment strategies. Thus, altered
expression of numerous genes in a different tissue was
observed, including lymphocytes. We suppose that using a microarray
approach in lymphocytes could be an attractive approach toward our
understanding of the molecular mechanisms of pathogenesis of RA
and will allow to identify potential targets for diagnostic
procedures and therapeutic intervention. SYSTEMIC LUPUS ERYTHEMATOSUS Molecular mechanisms SLE involves immune abnormalities in a wide variety of cell
populations including B and T lymphocytes, monocytes, and NK
cells. In murine models of SLE, an altered production of both Th1
(such as IFN- γ and IL-2) and Th2 (such as IL-4 and IL-10)
cytokines has been reported [ 82]. The disease activity in
animals significantly improved and/or the production of antibodies
decreased by treatment with anti-IFN- γ receptor
[ 83]. A similar effect was obtained with
anti-IL-4 [ 84] or IL-10 antibodies [ 85]. The entire
cytokine profile produced by circulating lymphocytes has as yet
not been clearly elucidated in human SLE. Production of
intracellular IL-10 was higher in B cells (predominantly in the
CD5 + cell subset) of SLE patients as compared to controls,
indicating its implication in the immune dysregulation in SLE
[ 86]. Serum IL-10 values seem to reflect SLE disease activity
and it has been suggested that overexpression of IL-10 might play
a pathogenic role [ 87] and that modulation of the level of
IL-10 may be of potential therapeutic benefit [ 88]. It has
been shown by Sturfelt et al [ 89] that relative absence of
IL-Ra response appears to be a feature characteristic of kidney
involvement in SLE patients. Imbalance between Th1 and Th2 cytokines appears also to be a
hallmark for SLE. Patients had increased levels of serum IL-4,
IL-10, IL-12, and IL-18. Moreover, IL-18 has been found to be
disease-activity related. A recent study has demonstrated that
IL-12 inhibits in vitro immunoglobulin G production in SLE
patients and reduces anti-dsDNA-secreting cells [ 90]. Several defects of T-cell signal transduction pathways have been
discovered over the last decade [ 91, 92]. They include
diminished T-cell receptor ζ-chain expression in T cells in
a majority of SLE patients withdefective
cAMP-dependent protein phosphorylation due to deficient activities
of type I and type II isozymes of proteinkinase A (PKA) [ 93].
Moreover, deficient PKA activity in SLE T cells may contribute in
part to impaired Ca 2+ homeostasis resulting in
calcineurin-catalyzed dephosphorylation of the transcription
factor NF-AT. Genes whose transcription is regulated by NF-AT,
such as CD154, Fas ligand, and c-myc, are overexpressed
in SLE T cells [ 94]. Also Georgescu et al [ 95] showed
increased spontaneous apoptosis of lymphocytes that has been
linked to increased IL-10 production, release of FasL, and
overexpression of the FasR in SLE. Abnormalities of two
transcription factors have recently been identified in SLE T
cells: (1) reduced/absent p65-RelA subunit of NF-kB and (2)
increased phosphorylated cAMP response modulator (p-CREM) binding
to the IL-12 promoter [ 96]. Changes in the NGF levels were found in plasma of adult patients
with SLE [ 97]. Already in childhood NGF levels can be
correlated with disease activity [ 98]. These results suggest
that NGF may play a role in the pathogenesis of SLE and that NGF
levels may be of prognostic value in evaluating the course of the
disease and outlining the medication. Gene expression Gene expression in lymphocytes showed high expression of IL-4,
IL-6, IL-10, and TNF- α in SLE patients as compared to
control [ 99]. The resulting high level of cytokines with
strong effect on proliferation and differentiation of B
lymphocytes could be responsible for characteristic B-cell
hyperactivity and autoantibody production seen in SLE. Results of
other investigations [ 100] demonstrated impaired production
of IL-12 in SLE lymphocytes and deficient IL-12 p40 gene
expression. Downregulation of IL-12 p40 gene expression appears to
be the cause of IL-12 p70 deficiency in SLE. IL-12 and
IFN- γ inhibit IL-10 expression and reduce IL-10-secreting
cells. This indicates that correction of the deficiency of IL-12
and IFN- γ in SLE may normalize pathogenically excessive
IL-10 and help remit the disease. Rus et al [ 8] using cDNA microarray in PBMCs of 21 SLE
patients and 12 controls demonstrated that of 375 potentially
relevant genes 50 genes exhibited more than 2.5-fold difference in
expression level compared to healthy control and twenty genes were
significantly different. Most of these genes have not previously
been associated with SLE and belong to a variety of families such
as TNF/death receptor, IL-1 cytokine family, and IL-8 and its
receptors. For five of the differentially expressed genes found in
this study, changes at the protein-expression level have been
previously reported in SLE. These investigators have recently
published expression pattern of 375 genes in PBMCs from 12
patients with active and 14 patients with inactive diseases; 29
genes were found to best discriminate. Among these genes, 14 were
upregulated and 15 were downregulated in patients with active
diseases compared to those with inactive diseases.
Most
of these genes have not been previously associated with disease
activity and belong to a variety of families such as adhesion
molecules, proteases, TNF superfamily, and neurotrophic factors
[ 101]. Very interesting results that immunosuppressive
therapy modulates T lymphocyte gene expression in SLE patient were
presented by Pereira et al [ 102]. They observed that
untreated patients have 38 repressed genes as compared
to healthy control. When untreated patients were compared to
treated ones, 154 genes were upregulated. Serum soluble receptor (srIL2f, p55 srTNFf, p75 srTNFf) levels
were higher in SLE patients with nephritis before treatment and
decreased significantly 6 month after treatment, suggesting that
soluble receptors of cytokines are sensitive markers of diseases
activity [ 103, 104]. SLE B cells have bone-resorbing activity
which corresponds to IL- α, and IL- α produced by B
cells might be one of the causes of bone destruction in SLE
patients which has also been reported [ 105]. In T cells from patients with SLE, activity of type 1 protein
kinase A isozymes is greatly reduced because of decreased
expression of the α and β regulatory subunits
(RI α and RI β). Mutations of the RI α subunit
were observed in T cells of patients with SLE, caused by
overexpression of an IFN- α-inducible transcript editing
gene, ADAR1 [ 106]. Gene-expression profiling with a microarray using PBMCs from SLE
patients and controls comprised of monocytes/macrophages, B and T
lymphocytes, and NK cells demonstrated expression of 4566 genes
represented on the chip. This analysis identified 161 unique genes
that were differentially expressed by the following criteria:
changes in expression of least 1.5-fold, and difference in
expression of at least 100 expression units when comparing the
means of two groups. Most of the genes that best distinguished SLE
from control PBMCs were more expressed in SLE (124 of 161,
77%). Many SLE patients were found to overexpress mRNA for the
cell surface markers: TNFR6 (Fas/CD95), a death receptor,
intercellular adhesion molecule-1 (CD54), a lymphocyte activation
antigen, and complement receptor. Other notable overexpressed
genes included the signaling molecules MAP3K-8, RAB27, and the
interleukin-6 signal transducer, and the transcription factors
v-ets 2, and others [ 107]. Increased IFN- γ, IL-10 and decreased IL-4 mRNA expression
in PMBCs from patients with SLE have been reported [ 108]. The
results of several investigations showed that IFN-regulated genes
are among the most significantly overexpressed in SLE mononuclear
cells [ 109]. The changes in gene expression after IFN treatment have recently
been investigated. This analysis identified 286 genes that
demonstrated > 2-fold change in expression from baseline, and
absolute mean difference in the level of expression > 500 units.
The induction of many IFN-regulated genes, such as STAT1,
myxovirus resistance 1(Mx-1), and ISGF-3, validated the approach.
Linear regression analysis showed that the IFN score was
significantly correlated with the number of SLE criteria, so an
elevated IFN score is strongly associated with the most severe
manifestation of SLE [ 107]. In addition, enhanced mutational
activity of immunoglobulin genes has been implicated in the
pathogenesis SLE [ 110, 111]. Although the causes of
autoantibody production have not been completely delineated, it
has been proposed that a failure of editing or revision of
autoreactive B-cell receptors contributes [ 112]. The
rearrangement of immune-receptor genes depends on recombination
activating gene (RAG)
enzymes [ 113]. A recent study of Girschick
et al [ 114] has showed that RAG expression is upregulated in
peripheral IgD+ and VpreB+ B cells of patients with active SLE.
These cells may contribute to the immunoregulatory abnormalities
in patients with SLE. Thus, there are several well-documented
disturbances of gene expression in lymphocytes in SLE. Therefore,
these cells have the potential for identification of genes
responsible for development and progression of SLE, prognosis, and
treatment strategies. Comparison of the expression profiles In Table 1, we summarize the expression profiles of
PMBCs during the diseased state of asthma, RA, and SLE. It can be
concluded that there is a significant overlap
concerning direct inflammatory markers, whereas of the others no
systematic investigations are known to conclude disease
specificity. Such data show that inflammation-related gene
expression is likely to be of little use for diagnostic purposes,
but may be well suitable to follow the time course of the 3
disorders. Methodological considerations Any method of isolation of white blood cells stimulates
lymphocytes and mRNA expression. Therefore, the method of
collection lymphocytes should be well standardized. In addition, a
lymphocyte activation procedure may be considered.
Accordingly, the expression of particular mRNA may thus become
increased several-fold allowing precise determination. We have
experience using Ca2+-ionophore to increase the expression of
transduction-associated genes several up to 100-fold (unpublished
data). It is therefore important to standardize
collection of PBMCs carefully, which may pose a logistic burden in
interinstitutional clinical investigations. In some cases, a
specific activation technique or circumstances may be necessary to
optimize gene expression. Gene expression requires specific
laboratory facilities, but in essence PBMCs can be recovered at an
extra-laboratory site and shipped afterwards to laboratory
facilities for activation and mRNA extraction. In this scenario,
there must be equipment available to isolate vital cells from
blood on location, including a cooled centrifuge. Then storage and
transport of vital cells become mandatory. In the last decade,
techniques have developed to keep cells vital for longer periods
(months) by controlled freezing (eg, −1°C per min) and
then storing them at −80°C. Although these logistic
problems can be overcome, most experiments will be designed in
such a manner that blood can be taken at a laboratory
facility. It is here not the place to discuss strong and weak points of the
microarray technique in detail. Excellent reviews
on technical aspects have appeared and sources are available
online at http://www.gene-chips.com/. So we are not discussing
the variations of the technique itself, but rather whether some of
the shortcomings of the techniques may be avoided or at least the
consequences of them reduced. The basis of
the microarray technique is those cDNA oligonucleotides
spotted on the array that are complementary to the mRNA
of the biological sample. But because of a
nonspecificity of the binding, there is a relatively large noise
and consequently a low signal-to-noise ratio. In
particular, when the levels of expression of the mRNA are low, the
general background fluorescence and nonspecific
binding of the labels mRNA's at the microarray may overshadow the
biologically significant signals. Amplification of the mRNA, to
reach higher levels of mRNA, is then considered. Such
amplification may, however, not always be linear and may
artificially increase or decrease the relative expression. Pooling
of mRNA extracts of various blood samples may indeed lead to
higher biological signals, but may also dilute the relevant
differences, unless done carefully. We will propose a procedure
for optimal pooling (see below). Finally, we would emphasize the potential of the microarray
technique to allow direct comparison of 2 samples with each other
on a single microarray. In that case, the
biomedical samples are stained with different fluorescent labels
(Cy3 and Cy5), so the ratio of the labels indicates the relative
expression of a gene. In such analysis, an ideal
competition between the differentially labeled mRNA's is assumed
to occur. This may, however, not be the case, as
labeling may influence binding properties. Such
complication may well be avoided by repeating the assay, but with
changing the labeling. If its results are consistent, it is
obvious that labeling did not influence the profiles. Another
concern may be the technique of averaging the signal. In most
procedures, the average fluorescence of the chip is
taken as an indication of the mean background. Such signal may,
however, be unevenly distributed over the array and in some cases
mean background of an area surrounding the genes of interest
is taken, which may comprise less than 10% of
the all detected gene products. Dedicated clinical designs Peripheral white blood cells (and lymphocytes) are easy to obtain
and express a substantial part of the human genome. Here we will
discuss some of the potentials and also pitfalls of the
current use of microarrays in clinical studies. It may be
emphasized here again that mRNA
differs from gene analysis as it shows state-dependent variations,
for example, it can be used to assess differences of gene
expression in the diseased and the recovered state of the same
subject. In the currently published studies, this unique
possibility has not well been exploited. State-of-the-art
microarrays allow detecting the whole genome (30 000 genes) and
may easily lead to several thousands of false positives that are
changes seen in cases as compared to the reference samples. In the
case with largely different expression, such as in tumor cells,
comparison with nonproliferating cell may suffice to discern
differences in expression. In the here considered use of
microarrays in clinical studies, a far more sophisticated approach
is required to obtain maximal benefit of the array
technology. Another relevant question is whether differential expressions
should be studied in pairwise or against a reference sample (or
standard). As described below, we prefer to compare the most
relevant samples directly, thus avoiding the use of a reference
mixture or sample. In current microarrays, the relative
differences in a single micro-array can be shown directly, for
example, as one sample is tagged with green fluorescence and the
other (eg, control) is provided with a red fluorescent label. It
should be noted here that the efficacy of the labeling may differ,
so that there is no guaranteed linearity between the green and red
labels. Moreover, each labeling is not always linear and the
linear range of Cy3 and Cy5 may differ. Current practice is to
label the mRNA extracts to be compared on a single array in
different proportions and with alternating label. Increase of the amount of mRNA increases the signal-to-noise ratio
of the array. Moreover, genes that are very well expressed
give—obviously—a better signal-to-noise ratio as well. The
relative differences in expression may well differ more than
1 000 000 for the genes. Pooling of samples may well increase
the sensitivity of the measurement, but still combining
10
blood samples the expression level may still remain low and
marginal. Pooling has, however, in addition to the possible
increased signal-to-noise ratio also the advantage that it may
eliminate some of the biological variance. For instance, consider
a noise of 100 and a net biologically relevant signal of 30, so to
conclude to a significant difference one must distinguish
100 from 130,
which falls below the current technical possibilities. If however
one pools 5 samples, then the difference between (relevant) signal
and noise is now 100 and 250, which is well detectable. But also
in an attempt to detect the differences between state 1 and state
2 of a subject, pooling of the extracts obtained at state 1 versus
those at state 2 may well be helpful. If, for example, the
relative alterations in gene expression are variable and small,
but the direction of change is similar, already pools of 5 samples
and 10 arrays may well allow the identification of a small number
of genes, characteristic for the disease state investigated. In this review, we summarize relevant investigations of the gene
expression in asthma, RA, and SLE, internal diseases with altered
immune response in which lymphocytes play a central role. In our
opinion, microarray studies in human lymphocytes would allow us to
monitor alteration in gene expression relevant to asthma, RA and
SLE and to possibilities of future therapeutically intervention.
Further characterization of the gene expression in lymphocytes
using cDNA microarray could help identify more precise
pathophysiological mechanisms in these disorders, and could find
beneficial therapeutic approaches. This suggestion has recently
been supported demonstrating coordinate overexpression of
interferon- α-induced genes using PMBCs as model in SLE
[ 115]. SLE is characterized by various alterations
in gene (and gene products) expression leading
to diverse dysfunctions of T cells, B
cells, and NK cells and as result development of clinical
symptoms. Our suggestions have also been supported by the very
recent report of Qing and Putterman [ 116]
from the 4th International Congress of Autoimmunity. Most of the genes involved in the pathogenesis of these diseases
are belonging to sets of signal transduction molecules,
inflammation-related cytokines (and chemokines),
apoptosis-inducing molecules, cell-cycle proteins, or
transcription factors. Most of these subsets of genes are now
commercially available to be used on various microarray systems.
The application of these systems has become more user-friendly and
less expensive lately, and approaches the point to be a common
tool available at most medicine departments. Alizadech et al
[ 117, 118] have developed a “lymphochip,” which is a
microarray of merely 10 000 individual human cDNA, representing
genes of known and unknown function expressed on lymphocytes. Such microarray technique gives the clues for identifying a novel
responsible genes which underlie the process of the disease, and
also could help identify appropriate targets for therapeutic
intervention. Beside this, the microarrays using a
“lymphochip” could be potential tools for investigating
the mechanism of drug action. The use of the microarrays does not
allow only comparison of the expression profile in different
subjects (for intersubject designs), but also in individuals
before, during, and after the disease (for intrasubjects designs).
Lymphocytes are an easily accessible model to be investigated with
microarray techniques. This approach is minimally
invasive, brings us valuable information at the cellular and
molecular level of the disease, and can be universally applied in
clinical medicine. There are, however, many potential pitfalls in
the use of microarrays that result in false leads and erroneous
conclusion [ 119]. In order to control the many sources of
variation and the many opportunities for misanalysis, DNA
microarray studies require careful planning, experimental design,
statistical analysis, and interpretation. Different studies have
different objectives, and important aspects of design and analysis
strategy differ for different types of studies. Studies of disease
with cDNA microarray technology can be split into two main
categories with interrelated goals: identification of key
molecular changes in diseases and identification of biomarkers or
molecular fingerprints that will aid in patient diagnosis and
classification. Studies that identify molecular changes in disease
will advance our understanding of disease pathophysiology, whereas
studies that identify biomarkers will improve diagnostic accuracy
and targeting of specific therapeutic interventions [ 120]. We
therefore suggest that exploring of the cDNA microarray gene
expression in blood lymphocytes could be an advantageous and
powerful tool for research, diagnostic, and treatment purposes in
internal medicine. The authors gratefully thank Professor D S Postma, Department of
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nonspecific gene expression,