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Analysis of Cytokine Expression in Dermatology
Khusru Asadullah, MD;
Wolfram Sterry, MD;
Hans-Dieter Volk, MD
Arch Dermatol. 2002;138:1189-1196.
ABSTRACT
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During the past decade, the detection of cytokines has been a focus
of scientific interest, including in dermatology. Dysregulation of cytokine
production seems to be involved in the pathogenesis of various diseases. The
determination of cytokine levels is of increasing diagnostic importance, and
cytokines are used as therapeutic agents too. Cytokines are polypeptides secreted
by a wide variety of cells in response to diverse stimuli, and mediate autocrine,
paracrine, or endocrine effects that are often pleiotropic and redundant.
Their molecular weight lies between 6 and 70 kd. The cytokines of immunologic
relevance are primarily those that are formed by immune cells (monokines and
lymphokines) and/or influence their function. In principle, cytokines are
detectable on 3 levels: (1) By using polymerase chain reaction, the messenger
RNA expression of cytokine genes can be detected and, with the newer techniques,
even quantified. (2) Protein synthesis can be detected by using bioassays
and enzyme immunoassays or immunocytologic or immunohistologic detection of
intracellular cytokine production. (3) Finally, there are indirect methods
for the detection of cytokine formation by analysis of products of cytokine
activity. The immunobiological features of cytokines and the different approaches
for cytokine determination are briefly discussed herein because basic knowledge
of these biologically highly active messenger substances and the capabilities
and limits of the individual detection methods are essential for a sensible
interpretation of the relevant findings.
INTRODUCTION
Cytokines play an essential role in the intercellular communication
network. They are essential for the mediation and regulation not only of inflammatory
reactions but also of specific immune reactions and nonimmunologic processes
(hematopoiesis and bone and cartilage metabolism). The importance of cytokines
in medicine is increasing dramatically. They are far beyond the stage when
they were of interest only to the research sector; cytokine therapy of various
diseases has long been used clinically.1 The
use of cytokine therapy in dermatology has recently been reviewed.2 Table 1
gives an overview. Moreover, the diagnostic determination of cytokine expression
is being used clinically as well. Before addressing the possibilities and
limits of the different approaches for cytokine determination, an overview
of nomenclature and receptor binding and signaling and their functional consequences
will be given.
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Table 1. Examples of Cytokine Therapy in Dermatology*
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NOMENCLATURE
The nomenclature of cytokines is confusing and has changed over time.
Initially, cytokines were generally given a name describing their function
(eg, tumor necrosis factor [TNF], which induces necrosis of some tumors).
However, it was soon discovered that the functional behavior of cytokines
is often redundant (several cytokines have the same effects) or pleiotropic
(one cytokine has several different effects) (Figure 1 and Figure 2,
respectively). Based on these facts, new cytokines are not named according
to their properties. An international nomenclature, at least for the immunoregulatory
cytokines, has been developed. The relevant cytokines were designated as interleukins
(ILs) and provided with numbers (currently, IL-1 to IL-24); the IL-1 receptor
antagonist is a relevant cytokine as well. However, numerous cytokines were
omitted from this nomenclature. They include interferons (IFNs) (IFN- ,
IFN- , and IFN- ), colony-stimulating factors (CSFs) (macrophage
CSFs, granulocyte-monocyte CSFs, granulocyte CSFs, stem cell factors, erythropoietin,
and thrombopoietin), TNFs (TNF-, TNF-, and lymphotoxin ),
chemotactic factors (macrophage inflammatory protein and monocyte chemoattractant
protein), and soluble receptors (CD23, p55–IL-2 receptor [IL-2R], IL-4
receptor, TNF receptor, and IL-1 receptor).
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Figure 1. Examples of the redundancy of
cytokines. IFN indicates interferon; TNF, tumor necrosis factor; and IL, interleukin.
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Figure 2. Pleiotropic activity of tumor
necrosis factor (TNF) . Plus sign indicates induction; minus sign,
inhibition; CNS, central nervous system; PGE2, prostaglandin E2; and IL, interleukin.
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The diversity of cytokine effects makes a didactically sensible nomenclature
difficult. In principle, cytokines can be divided into 3 groups, based on
their origin and effect: (1) those produced by many cell types that act on
many cell types (eg, transforming growth factor ); (2) those produced
by few cell types that act on few cell types (eg, IL-2); and (3) those produced
by few cell types that act on many cell types (eg, TNF- and IFN-).
From the viewpoint of their effect on inflammation processes, cytokines
can be divided into proinflammatory (eg, IL-1, IL-2, INF-, and TNF-)
and anti-inflammatory (eg, IL-1 receptor antagonist, IL-10, and transforming
growth factor ). Certain soluble receptors are naturally occurring
antagonists of cytokines.
IMMUNOBIOLOGICAL FEATURES
The cytokines of immunologic relevance are primarily those that are
formed by lymphocytes and antigen-presenting cells and influence their function.
For many years, cytokine production by lymphocytes has been the object of
intense investigation. Based on the results of a study by Mosmann et al,3 published in 1986, murine CD4+ T cells
can be divided into subclasses. Helper T (TH) 1 cells are characterized
by the production of IL-2, TNF-, and INF-, whereas Th2 cells
produce mainly IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Whether a TH0 cell (a cell with no preferred cytokine secretion pattern) differentiates
into a TH1 or a TH2 cell under antigen influence depends
essentially on the local cytokine milieu. Interleukin 12 and INF- encourage
development of the TH1 cell response, whereas IL-4 favors the TH2 cell response (Figure 3).
An essential role in the origin of the local cytokine milieu is played by
non–T cells too (eg, natural killer cells [INF-], monocytes or
macrophages [IL-12 or IL-10, according to stimulus], and mast cells [IL-4]).
CD8+ T cells usually secrete a TH1 cell–like pattern.
Consequently, because not only CD4+ TH cells but also
other cell populations contribute to local cytokine expression, the cytokine
pattern is often designated as type 1 or type 2 accordingly.
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Figure 3. Helper T (TH) 1/2 cell
balance. The development of TH1 or TH2 cells is influenced
by several factors. Whereas a TH1 phenotype predominates in patients
with psoriasis and allergic contact dermatitis, a TH2 phenotype
predominates in patients with atopic dermatitis and late-stage cutaneous T-cell
lymphoma. MHC indicates major histocompatibility complex; IL, interleukin;
and IFN, interferon.
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The TH1/TH2 or type 1/2 cytokine model has made
a major contribution to our understanding of immunologic processes. To simplify,
one may say that a type 1 cytokine pattern is necessary for an effective cellular
immunologic reaction to antigen, such as the immune response to intracellular
pathogens,4 although the secretion of immunoglobulin
subclasses IgG1 and IgG3, which have a high capacity to interact with immune
cells via Fc and C receptors, is stimulated by type 1 cytokines too. Type
2 cytokines, on the other hand, are mainly responsible for effective humoral
immunologic mechanisms. Thus, these cytokines support IgE, IgA, IgG2, and
IgG4 synthesis and eosinophil and mast cell growth. Consequently, they play
a particularly decisive role in defense against intestinal parasites, neutralization
of bacterial toxins, and local mucosal defense.5
Type 1 and type 2 responses are essential for an adequate immunity.
In recent years, cytokine patterns have been investigated in diseases
from nearly all specialties. This has made a major contribution to today's
better understanding of pathophysiological processes. An attempt has been
made to identify classic type 1 or type 2 diseases. In fact, in many diseases,
an immune deviation toward a type 1 or a type 2 cytokine pattern has been
observed and might be of pathophysiological relevance5
(Figure 3). However, the limitations
of the type 1/type 2 system are becoming increasingly clear and prevent dogmatic
application. Thus, several investigations6
have revealed a remarkable variety of cytokine patterns, indicating a broad
spectrum of cytokine expression in which the classic type 1 and type 2 patterns
are merely the extremes. Moreover, the existence of another important T-cell
population with regulatory properties (T-regulatory and TH3 cells)
has been demonstrated recently.7-8
These cells predominantly act immunosuppressively via secretion of cytokines
like IL-10 and transforming growth factor and via cell-cell interaction.
Meanwhile, the complexity of cytokine expression has also been demonstrated
in vivo—taking dermatology as an example—in patients with psoriasis,9 atopic dermatitis,10
and mycosis fungoides.11 For example, stage,
dependent shifts (for atopic dermatitis, early phase type 2 and late phase
type 1), and untypical cytokine patterns (the advanced stage of mycosis fungoides
is like type 2 but shows weak IL-4 expression) can be observed.
CYTOKINE RECEPTORS AND SIGNAL TRANSDUCTION
Ligands and Receptors
Class 1 receptors bind factors designated variously as ILs, hormones,
or CSFs (Table 2), but, despite
the different designations, the ligands all have a common 4-–helical
structure.12-13 These receptors
are transmembrane proteins. Intracellularly, they have a conserved membrane–proximal
domain that serves to bind Janus kinases (Jaks).14
The conserved features of the ligands and receptors permit computational database
mining and the recent identification of new family members.15-17
Class 2 receptors are structurally related to class 1 receptors and bind IFNs
and IL-10 family cytokines; this family is rapidly expanding.18
Recently, researchers discovered the first soluble class 2 receptor.19
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Table 2. Types of Cytokine Receptors and Use of Jaks and Stats by Various
Cytokines*
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Sharing of receptor subunits is an important feature of cytokines binding
class 1/2 receptors that defines subfamilies (Table 2). 12 For instance, IL-2, IL-4,
IL-7, IL-9, and IL-15 use the common chain in conjunction with ligand-specific
chains, which explains the specific and redundant actions of these cytokines.
Moreover, some cytokines, like IL-2 and IL-15, share even 2 receptor subunits
(IL-2R and the common chain), but have an additional ligand-specific
binding subunit (IL-2R or IL-15 receptor ). Another level of
complexity is that more than one type of receptor may exist, as in the case
of IL-4. One IL-4 receptor consists of the IL-4 receptor associated with the
common chain, whereas a second comprises IL-4 receptor with IL-13
receptor. Finally, a third level of complexity has been illustrated by IL-12
and IL-23.16 The p40 subunit is not homologous
to other cytokines but rather to cytokine receptors. It was first identified
in association with p35 to form IL-12. In contrast, IL-23 comprises p40 and
a novel subunit, p19; the latter is homologous to other cytokines. Like IL-12,
IL-23 binds to the IL-12 receptor 1 chain and induces IFN- production.
However, IL-12 induces IFN- in naive and memory T cells, and IL-23
acts on memory T cells only. This may be because IL-23, in contrast to IL-12,
does not bind to the IL-12 receptor 2 chain.12
Intracellular Signaling
The action of cytokines is not exclusively regulated by the level of
ligand and receptor expression, but is also dependent on intracellular signal
transduction. At least 3 classes of molecules are of major importance: classic
transcription factors (eg, nuclear factor  B and activator protein 1),
Jaks, and signal transducers and activators of transcription (Stats). Moreover,
other intracellular proteins are involved in determining the biological consequences
of cytokine presence. Therefore, 3 families of negative regulatory proteins
have been discovered: phosphatases, protein inhibitors of activated Stats,
and suppressors of cytokine signaling. Each of these protein families seems
to act at a distinct point and at a particular time in the cytokine signaling
cascade. It is important to be aware of this high complexity when interpreting
results from cytokine expression analyses.12
The Jaks constitutively bind the membrane-proximal domains of cytokine
receptors and seem to be the major initiators of signaling (Table 2). Four mammalian Jaks have been identified—Jak1, Jak2,
Jak3, and Tyk2 (a small family of tyrosine kinases with rather specific functions,
as illustrated by gene-targeted mice). After activation, the Jaks phosphorylate
receptor subunits on tyrosine residues to recruit proteins with src homology
2 domains or phosphotyrosine-binding domains. These proteins, in turn, are
also phosphorylated by Jaks, coupling cytokine stimulation to several pathways,
including the Ras/Rat/MAPK and phosphatidylinositol 3' kinase/Akt pathways.12 Phosphorylation of cytokine receptors also generates
docking sites for a class of src homology 2 domain–containing cytosolic
molecules termed "Stats."20-23
Receptor-bound Stats are then phosphorylated (Table 2), allowing them to dimerize. This permits translocation
to the nucleus and DNA binding. Generally, Stats bind 2 types of DNA motifs:
IFN-stimulated response elements and -activated sequence elements.
For Stat6, there seems to be little specificity in Stat binding to DNA. The
other Stats seem to be a relatively small family of transcription factors
with highly specific functions; remarkably, it seems that at least 4 of the
6 mammalian Stats have major functions in regulating host defense and immune
responses. There is only limited knowledge about cytokine-inducible genes
and how Stats work with other transcription factors to regulate the expression
of these genes.12 The transcription factor
nuclear factor B is a main mediator of many proinflammatory cytokines
(TNF- and IL-1). Further investigations, in particular on the role
of negative regulatory proteins (eg, suppressors of cytokine signaling), will
be important if the physiological and pathophysiological relevant controls
on cytokine responses are to be uncovered.
DETECTION OF CYTOKINES
Cytokines exert their biological effects at concentrations in the picomolar
range. The biological half-life in vivo is short, generally not exceeding
3 minutes in plasma. An exception is IL-12, whose half-life is several hours.
The short in vivo half-life is the result of binding to cells (high-affinity
receptors) or plasma proteins, proteolytic degradation, and elimination via
the kidneys. Moreover, cells nearly always produce cytokine for only a few
hours after appropriate stimulation. This results in limited systemic availability,
which makes relevant quantitation in plasma difficult. The physiological task
of cytokines consists principally in the regulation of local intercellular
communication processes, ie, the effects are largely of an autocrine or paracrine
nature, whereas endocrine effects are fairly rare.
In principle, a distinction can be made between in vivo, ex vivo, and
in vitro cytokine detection. A positive finding in in vivo determinations
indicates that the cytokine is actually present, whereas ex vivo and in vitro
methods (such as the whole-blood assay24-25)
use stimulants and primarily reflect the cytokine expression capacity of the
stimulated cells and not necessarily the actual activity in vivo. Endotoxin,
for example, is often used as an ex vivo stimulus for monocytes and macrophages.
Lectins, anti–T-cell antibodies, or superantigens are used as T-cell
mitogens. Cytokine detection can take place on various levels (Figure 4).
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Figure 4. Cytokine detection options on
various levels. mRNA indicates messenger RNA; ELISA, enzyme-linked immunosorbent
assay.
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Cytokine Detection at the Gene Expression Level (Messenger RNA)
These determinations are based on the observation that resting immunocytes
express little or no cytokine messenger RNA (mRNA). Within a few hours after
stimulation, an increase in mRNA level is seen. Whereas the sensitivity of
conventional detection methods is sufficient to detect the mRNA of typical
inflammatory cytokines (eg, TNF, IL-6, and IL-8) without amplification of
the specific nucleic acid sequence (eg, in situ hybridization and a nuclease
protection assay), the mRNA expression of most T-cell cytokines (eg, IL-2
and IL-4) is barely detectable with these methods in in vivo samples. A much
higher sensitivity is achieved by cytokine mRNA detection using the polymerase
chain reaction (PCR) (Table 3).
Nevertheless, quantitation of the degree of expression is much more difficult
than in the hybridization procedures without prior amplification of complementary
DNA (cDNA). Competitive reverse transcription–PCR,26
in which a DNA competitor fragment added in a known concentration competes
with the target cDNA for the primer, is suitable for quantitative determination.11, 27-28 However, real-time
reverse transcription–PCR is the most reliable PCR method. It uses the
5' nuclease activity of Taq polymerase to cleave
a nonextendable hybridization probe during the extension phase of the PCR.
The approach uses dual-labeled fluorogenic hybridization probes. One fluorescence
dye serves as a reporter, and its emission spectrum is quenched by the second
fluorescent dye. Following hybridization of the labeled probe, reporter and
quencher are separated, resulting in fluorescence. The reactions are monitored
in real time during the log phase of product accumulation. The increase of
the reporter dye fluorescence intensity during PCR is proportional to the
amplification of the target sequence. The cycle number at which the amplification
plot crosses a fixed threshold above baseline is defined as the threshold
cycle. These values can be used for comparison of mRNA content. To control
variation in cDNA contents throughout different preparations, all results
are related to the concentration of a gene whose expression remains relatively
constant ("housekeeping gene" [eg, the human hypoxanthine-phosphoribosyl-transferase
gene]).29
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Table 3. Cytokine Detection on the Gene Expression Level (mRNA)*
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When using a PCR method to investigate cutaneous cytokine expression,
one obvious disadvantage is the lack of information regarding the cellular
source of a cytokine found to be overexpressed. Drawing conclusions from the
cellular pattern determined simultaneously by histologic investigation requires
caution, because small cell numbers can also be responsible for large amounts
of cytokines, whereas even high numbers of certain cells types could be present
without producing any cytokines. To determine the compartment mainly responsible
for the cytokine expression, splitting skin samples and comparing epidermis
with dermis can be considered. Such analyses, however, are usually not sufficient
to identify the cell type because of the complex cellular composition of epidermis
and dermis, which is even more complex under pathophysiological conditions.
Also, splitting skin samples may affect the transcripts.30
Therefore, we prefer to analyze whole skin punch biopsy specimens immediately
shock frozen after attainment, without any further manipulation.
Hybridization by Northern blotting is a technique less sensitive than
PCR but enables the detection of splice variants. Gene chip analysis, in turn,
as a modern but expensive recent hybridization technique, has the advantage
of delivering information on the expression of up to 12 000 gene products
simultaneously (Table 3).
However, although cytokine gene expression is a prerequisite for protein
synthesis, it does not necessarily lead to such synthesis because there are
still several posttranscriptional regulatory bases. Hence, the detected cytokine
gene expression and the protein synthesis do not necessarily correlate with
each other. Examples of significantly posttranscriptionally regulated cytokines
are IL-1 and IL-18.
Cytokine Detection at the Protein Level
Historically, cytokines were initially detected solely with bioassays
(Table 4). For example, IL-2 was
detected based on its property to stimulate the proliferation of activated
T cells in vitro. By adding known quantities of IL-2, a calibration curve
can be plotted. This test is still used, using highly sensitive T-cell lines
whose growth depends on IL-2. However, its specificity is known to be limited
because other cytokines (eg, IL-4 and IL-7) mediate similar effects in this
test. This can be differentiated by adding a blocking IL-2R antibody. The
redundancy of cytokine effects must be borne in mind in other bioassays too.
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Table 4. Cytokine Detection on the Protein Level*
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Thanks to the use of internationally harmonized cytokine standards and
the distribution of correspondingly sensitive cell lines via international
tissue banks, the standardization of the bioassays has improved considerably.
Even so, the specificity of any detected bioactivity must always be checked
by adding specific neutralizing antibodies. Although bioassays are cheaper
than enzyme-linked immunosorbent assays (ELISAs) (detailed later), their high
intra-assay and interassay variance makes them unsuitable for clinical monitoring.
In recent years, numerous ELISAs for the detection of a wide range of
cytokines have been developed (Table 4).
Although they were initially less sensitive than bioassays, the new generation
of ELISAs is actually more sensitive than bioassays. Thus, most cytokines
are detectable even in the plasma of healthy volunteers. The high specificity
of ELISAs and the better standardizability are further advantages to be weighed
against the high costs. A limitation of ELISAs that is often overlooked in
their clinical evaluation is the fact that only results obtained with the
same test are generally comparable. This may result from the use of differing
standards, epitope specificity (and sometimes also the affinity) of the antibodies
used in the test, and the particular properties of the cytokines. Cytokines
do not only occur in free and native form but also bind to numerous carrier
proteins and can undergo rapid proteolytic cleavage. A wide variety of cells
secrete soluble cytokine receptors after activation. These generally have
neutralizing properties. On the other hand, they may prolong activity by inhibiting
cytokine elimination. Cytokines can also bind to other plasma proteins. Such
proteins cover certain epitopes on the cytokine surface that may or may not
still be recognized, depending on the epitope specificity and affinity of
the cytokine-complex antibodies used in the ELISA (Figure 5).
The interpretation of cytokine concentrations in body fluids (plasma,
urine, and lavage fluid) poses additional problems besides those just mentioned.
Storage of the samples can have a considerable influence on the results. False
high concentrations can arise from activation following coagulation and contact
with the syringe material. To avoid these difficulties, it is advisable to
(1) determine cytokine levels in plasma, preferably with calcium and magnesium
withdrawal (using EDTA or citrate instead of heparin, but some assays do not
work in the presence of EDTA), and (2) separate the cells and fluid rapidly
(<2 hours) by centrifugation. False low values are caused by proteolytic
degradation of cytokines ex vivo. This can be prevented by cold storage, enzyme
blockade, and rapid workup of the sample (freezing the cell-free supernatants
or performing the test within 120 minutes if possible). This makes it difficult
to send samples to central laboratories (worked-up samples on dry ice). The
various cytokines certainly differ in sensitivity. A further important aspect
is the expression of the concentrations. In plasma, this is unproblematic,
but in other body fluids, it poses a relevant problem. Quoting the urinary
concentration of a cytokine in picograms per milliliter makes little sense.
Similar considerations apply to other body fluids or when analyzing cytokine
concentrations in fluid obtained from cutaneous suction blisters. Moreover,
this iatrogenic tissue damage, by inducing the blister itself, certainly leads
to an immune response associated with cytokine release. Consequently, the
cytokine's pattern detected in suction blisters does not necessarily reflect
that what is actually present. However, for comparison between different groups
of patients in one trial using this technique under exactly the same conditions,
it could be a valuable method. In view of the instability of cytokines, however,
all portions would have to be frozen immediately.
Even while remaining alert to the previously mentioned methodological
problems, however, one must not forget that cytokines are formed only temporarily
and have an extremely short half-life in vivo. Although high values are informative,
low values usually tell little. It may simply be that the time at which the
sample was taken was unfavorable.
Another important method is the detection of cytokine-producing cells
by immunocytology or immunohistology. Tissue samples or cells from fluids
(blood, cerebrospinal fluid, and fine-needle aspirates) are fixed on slides,
made permeable (not always), and incubated with specific anticytokine antibodies.
Cytokine-producing cells are detected using immune enzyme methods (alkaline
phosphatase–anti-alkaline phosphatase or the peroxidase technique).
Immunohistochemical analyses are often used for analyzing frozen skin biopsy
specimens. However, unspecific staining (a false-positive result) is a frequent
problem, and antibodies could be washed out during the procedure, leading
to false-negative results. Moreover, cytokines are frequently detected at
an intercellular, not an intracellular, level.31-32
Therefore, one advantage of immunohistochemical analyses, the reliable identification
of the cellular source, is not always given, although it might be possible
in some cases.33 Test kits that permit intracellular
cytokine detection using flow cytometry are increasingly becoming available.
This technique is well established for analyzing blood cells. Recently, researchers34 developed methods that also allow the flow cytometric
determination of intracellular cytokine expression in mechanically disaggregated
cutaneous cells. Finally, Western blotting is still a valid technique for
detecting cytokines from tissue or cell lysates, but may not be as sensitive
as ELISA (Table 4).
Indirect Cytokine Detection (Products of Cytokine Activity)
There are several types of cytokine-induced products: soluble surface
molecules, including receptors (eg, soluble IL-2R), and adhesion molecules
(eg, soluble endothelial leukocyte adhesion molecule 1); soluble factors,
including neopterin and C-reactive protein; and cell-bound surface molecules,
including HLA-DR and adhesion molecules.
Starting from the thesis that only the effects of cytokines are of any
biological relevance, one approach for detecting cytokine production in vivo
relies on the measurement of cytokine activity. For example, IFNs induce the
release of neopterin from monocytes and macrophages. Neopterin has a longer
plasma half-life than cytokines and is excreted via the kidneys. Hence, determination
in plasma or urine reflects IFN synthesis in vivo. Neopterin can be quantified
using commercially available immunoassays.
Many cytokines induce the synthesis of adhesion molecules and HLA molecules
in immunocytes and nonimmunologic cells (eg, endothelial and epithelial cells).
These molecules can be easily detected by immunocytologic and immunohistologic
methods. However, the increased expression of adhesion or HLA molecules on
the cells following the action of cytokines is also associated with increased
release of such molecules by the activated cells. Soluble adhesion or HLA
molecules can then be measured by ELISA in plasma or other body fluids. Owing
to their more persistent production and longer plasma half-life, these cytokine-induced
molecules are easier to detect than the cytokines themselves. In view of the
redundancy of the cytokine effect (Figure
1), however, no exact correlation between the induction of such
a molecule and the production of a specific cytokine is possible.
PERSPECTIVE
More than 10 years of intensive cytokine research in clinical samples
has opened new insights into the pathogenesis of many diseases. The techniques
for detecting cytokines at the mRNA and protein level have been significantly
improved. Most important was the standardization of methods. Continuing with
such processes will be of crucial importance.
Modern (semi) automatic ELISA systems allow precise quantification of
cytokines in the picomolar range, with interassay and intra-assay variance
of less than 5% to 8%. Using standardized (semi) automatic systems for cytokine
detection and cell culture, functional assays allow a high level of reproducibility
(eg, for ex vivo lipopolysaccharide-induced TNF- release: <20% variance
during a 1-year follow-up in healthy patients). The introduction of the real-time
PCR and the cDNA array technology is a revolution for quantifying cytokines
at the mRNA level. The detection of cytokine mRNA by real-time reverse transcription–PCR
is faster and more precise (<20% variance) than traditional techniques.
Array technologies allow the simultaneous determination of several gene expression
products (gene chips) and, more recently, of proteins (protein-binding arrays)
in individual samples. The future will be the consequent use of standardized
automatic systems only. This will also include methods for RNA extraction
and cDNA synthesis, because sample preparation seems to become the bottleneck
for quality and quantity of analyzing cytokine mRNA expression. Further emerging
technologies may allow reliable detection of cytokine-producing cells (eg,
in situ PCR) or quantification without prior amplification (direct mRNA quantification
[eg, the branched DNA method]).
The new technologies will provide a better opportunity for well-designed
multicenter trials, which are necessary to evaluate the clinical relevance
of cytokine diagnostic methods. Although this will be associated with significant
costs, it may lead to major progress. For example, based on the expression
pattern of cytokines, it might become possible to determine distinct immunologic
subpopulations within one disease, such as psoriasis. This is not just of
academic interest. We learned that typically only subgroups of these patients
respond well to each of the multiple novel immunotherapies, which are to be
launched soon.35 It would be highly desirable
(and cost reducing) to identify patients who are likely to respond to each
approach before or during the initial phase of therapy.
AUTHOR INFORMATION
Accepted for publication April 19, 2002.
We thank Ulrich Zuegel, PhD, for preparing Figure 3; Sabine Goerlich and Garry Willett for editing the manuscript;
and Wolf-Dietrich Doecke, MD, for helpful discussions.
Corresponding author and reprints: Khusru Asadullah, MD, Corporate
Research Business Area Dermatology, Schering AG, Müllerstrasse 178, D-13342
Berlin, Germany (e-mail: khusru.asadullah{at}schering.de).
From Corporate Research Business Area Dermatology, Schering AG (Dr
Asadullah), and the Departments of Dermatology and Allergology (Dr Sterry)
and Medical Immunology (Dr Volk), University Hospital Charité, Berlin
Humboldt University, Berlin, Germany.
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