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Echinacea and anti-inflammatory cytokine responses: Results of a gene and

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Pharmaceutical Biology, 2009; 47(6): 500–508

RESEARCH ARTICLE

Echinacea and anti-inflammatory cytokine responses:

Results of a gene and protein array analysis
M. Altamirano-Dimas, M. Sharma, and J.B. Hudson
Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, Canada

Abstract
Preparations of Echinacea (Asteraceae) are frequently consumed for the control and prevention of
­rhinovirus-induced colds and other respiratory disorders. Since it is now generally believed that the symp-
toms of rhinovirus colds are due to the enhanced secretion of inflammatory chemokines and cytokines, we
decided to analyze the effects of rhinovirus infection and Echinacea treatment [defined extracts of E. purpu-
rea (L.) Moench] on cytokine/chemokine gene expression and protein secretion in a line of human tracheo-
­bronchial epithelial cells. Among the collection of more than 50 cytokines and chemokines present in the
gene arrays, 12 showed significant induction of expression by the virus (> 2-fold), some of them by more
than 5-fold. However, not all of these resulted in similar changes in the corresponding proteins, presum-
ably as a consequence of post-transcriptional changes. A total of 16 cytokines, mostly chemokines, showed
substantial protein increases, including several, such as the well known pro-inflammatory cytokines IL-6
and IL-8 (CXCL8), which were induced in the absence of additional transcription. These results support
the concept that virus-induced multiple inflammatory cytokines are responsible for the cold symptoms.
In most cases, one or both Echinacea preparations reversed the viral stimulation, thus providing a basis for
the anti-inflammatory properties attributed to Echinacea.
Keywords:  Echinacea; rhinovirus; common cold; chemokines; cytokines; anti-inflammatory; gene arrays;
cytokine antibody arrays

Introduction pro-inflammatory cytokines (Message & Johnston, 2004;

Echinacea (Asteraceae) is one of the most popular
herbal medicines for the prevention and treatment
of colds and flu, and has often been advocated for the
alleviation of asthmatic and other respiratory conditions
(Barnes et al., 2005; Schoop et al., 2006; Shah et al., 2007).
However, although more than 100 common cold viruses,
the rhinoviruses, have been isolated and characterized,
research has shown that these viruses cause little or no
cytopathology in nasal tissues in vivo or in epithelial cell
lines of nasal or tracheo-bronchial origin (Gwaltney,
2002; Mosser et al., 2005). These observations have led
to the belief that the familiar symptoms of a common
cold, the running nose, stuffiness, sneezing, sore throat,
and mucus membrane irritation, result indirectly from
the ability of these viruses to stimulate the secretion of
Sharma et al., 2006). Evidence also indicates that rhino-
viruses can play a role in exacerbating asthmatic, aller-
gic, and other chronic respiratory conditions (Johnston,
2005; Schaller at al., 2006; Wark et al., 2007; Xatzipsalti
et al., 2007; Hansbro et al., 2008).
The cytokines comprise a large family of multi-func-
tional proteins, which can be produced and secreted
by many cells, including epithelial cells, in response
to stimuli such as rhinoviruses and other respiratory
viruses (Message & Johnston, 2004; Schaller et al., 2007).
Among this class of proteins is a group of smaller pro-
teins, the chemokines (chemo-attractant cytokines),
which share a common property of attracting inflam-
matory cells, usually neutrophils, eosinophils, or vari-
ous T-lymphocytes, to the site of an infection or wound
(Balestrieri et  al., 2008; Vandercappelen et  al., 2008).

Address for Correspondence:  Dr. J. B. Hudson, Pathology and Laboratory Medicine, C-360 Heather Pavilion, 2733 Heather Street, Vancouver, British
Columbia, Canada, V5Z 1M9. Tel./fax, 1-604-875-4351; E-mail: jbhudson@interchange.ubc.ca
(Received 20 January 2009; accepted 20 January 2009)
ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK Ltd
DOI: 10.1080/13880200902839525 http://www.informapharmascience.com/phb

More than 50 chemokines and 18 chemokine receptors
have been identified in humans (Colobran et al., 2007;
Balestrieri et al., 2008), and their inflammation-related
functions appear to be quite redundant.
We decided to make a detailed analysis of gene expres-
sion in rhinovirus infected epithelial cells, by means of a
combination of cytokine gene arrays and corresponding
protein antibody arrays. Both approaches are necessary to
obtain a complete picture since it is well known that gene
expression can be controlled at various post-transcription
and post-translation stages (Fan et al., 2005; Proost et al.,
2006; Gomez-Martin et al., 2008). Consequently, changes
in transcription of cytokine genes might not be reflected
in corresponding changes of the functional proteins. Our
hypothesis was that rhinovirus infection would activate or
enhance the expression and secretion of specific inflam-
matory cytokines or chemokines, and that these events
could be reversed by Echinacea treatment.
Materials and methods
Echinacea extracts
Two commercial preparations (E1 and E2) were
obtained from North American commercial sources
and analyzed for their major constituents. These are
the same preparations used in our previous antiviral
studies (Vimalanathan et al., 2005). E1 was an aqueous
expressed juice extract of the aerial parts of E. purpurea
[Echinacea purpurea (L.) Moench, University of Ottawa
Herbarium accession number UO 19180]. The total
extractable polysaccharide content of the pressed juice
product was 23.7% w/w. After hydrolysis the monosac-
charide content was determined and found to be con-
sistent with a mixture of starch and Echinacea hemicel-
luloses and AGP (arabinose 8%, galactose 17.1%, glucose
25.4%, mannose 46.7%, rhamnose 2.3%, xylose 2.3%).
E2 was a 50% ethanol tincture, derived from E. pur-
purea roots (1:9 w/v) (University of Ottawa Herbarium
accession number UO19181). This preparation was ana-
lyzed by HPLC as described previously, and showed a
typical combination of caffeic acid derivatives and alky-
lamides (Binns et al., 2002). Both extracts were diluted in
cell culture medium and stored frozen at -30°C.
Extracts E1 and E2, at the concentrations used, did
not show any cytotoxic effects, or other microscopically-
visible effects, on the cells. They were devoid of endo-
toxin activity, as determined by means of a commercial
endotoxin test kit (Lonza).
Cell culture and virus
BEAS-2B, a human bronchial epithelial cell line, and the
rhinovirus-sensitive H-1 derivative of HeLa cells, were
Echinacea: Anti-inflammatory genes and proteins   501
both obtained from American Type Culture Collection
(ATCC, Rockville, MD). The standard growth medium
was Dulbecco’s MEM with 5-10% fetal bovine serum
(endotoxin-free). All culture reagents were obtained
from Invitrogen (Ontario). For experimental purposes
BEAS-2B cells were used, at 90-100% confluency,
between passages 25 and 40. All cultures were main-
tained in a 5% CO2 : 95% air atmosphere, at 37°C or, in the
case of RV-infected cultures, 34°C. Rhinovirus type 14
(RV 14) was obtained from ATCC, and was propagated
and assayed, by plaque formation, in monolayers of H-1
cells, as described previously (Sharma et al., 2006).
Infection and treatment of BEAS-2B cells
BEAS-2 cells were grown to 90%-100% confluency in
75 cm2 flasks. Medium was removed and replaced with
2.0 mL of fresh medium without serum, containing the
appropriate amount of stock RV (moi = 0.4 pfu/cell), or
medium only. The flasks were incubated on a shaker plat-
form at 34°C for 1 h. Unadsorbed virus and media were
removed and the cells washed once with 5 mL medium
without serum. Fresh medium was then added, 10 mL
per flask, containing where appropriate 100 µg/mL
of E1 or 50 µg/mL of E2. These concentrations were non-
cytotoxic (Sharma et al., 2006).
DNA microarray analysis
Complete details are described elsewhere (Altamirano-
Dimas et al., 2007). Briefly, total RNA was extracted from
cells 18 h after infection, by the Trizol technique, and
purified from uninfected and RV14-infected BEAS-2B
cells with or without extract E1 or E2. The mRNAs were
reverse transcribed into Cy5 (cyanin dye #5) labeled
cDNAs, and hybridized, together with Cy3 (cyanin dye
#3) labeled UHR cDNA (derived from Universal Human
Reference RNA), with DNA microarrays (Prostate
Research Centre, Vancouver Hospital) containing a total
of 13,816 human gene-specific oligomers, plus 536 con-
trols, in duplicate.
Each experimental sample was compared directly
with the reference sample on the same array. The arrays
were scanned after hybridization, and the data normal-
ized and statistically analyzed as described below.
Statistical analysis of arrays
The arrays were scanned after hybridization with
the ScanArray analizer laser scanner (Perkin Elmer).
Independent grayscale images, 16-bit TIFF images, were
generated for each pair of samples to be compared.
These images were analyzed to identify the arrayed spots
and to measure the relative fluorescence intensities for
each element. Signal median intensities and background
502   M. Altamirano-Dimas et al.
correction were quantitated using the computer program
Imagene 5.6. Signal intensities of each one of the 28,704
spots in the microarray were plotted. Only experiments
in which 80% of the spots were present and the ratio of
signal to noise was higher than 3 in at least 40% of the
total number of spots were selected.
Gene expression was evaluated with the aid of
GeneSpring 7.3.1 program. The intensity of every spot
in the microarray was compared with the intensity of
the Universal Human Reference RNA (two color experi-
ments) in order to obtain a signal ratio. Normalization
was performed for each spot. Normalized data were
subjected to statistical analysis of variance (ANOVA).
The procedure one way ANOVA was used to filter
out genes that did not vary significantly across different
groups with multiple samples. This allowed us to find
those genes that exhibited important changes between
various conditions of the experiment. This comparison
was performed for each gene, and the genes with suf-
ficiently small p-values were returned. Comparisons
were performed using parametric methods, variances
not assumed equal (Welch ANOVA) and p-value cut-off
at 0.05.
Clustering algorithms were used to divide genes into
groups that have similar expression patterns. K-means
clustering was used to divide genes into groups based
on their expression patterns. The goal was to produce
groups of genes with a high degree of similarity within
each group and a low degree of similarity between
groups. K-means clusters were constructed so that the
average behavior in each group was distinct from any of
the other groups.
Principal component analysis (PCA) was used as
a decomposition technique that produced a set of
expression patterns known as principal components.
Linear combinations of these patterns were assembled
to represent the behavior of all of the genes in a given
data set.
The phylogenetic tree or dendrogram was constructed
in order to view the results of hierarchical clustering. In
such a tree, genes having similar expression patterns
were clustered together.
The pathway views were used to display and place
genes on an imported .gif or .jpeg image. We used the
biochemical pathways imported from the database
Kyoto Encyclopedia of Genes and Genomes (KEGG)
and gene maps from GenMapp2 Pathways to see the
expression values in relationship with other proteins in
a pathway.
The normalized values obtained from GeneSpring
analysis were used to generate the normalized ratios
of E1/C, E2/C, RV/C, RVE1/RV, RVE2/RV and loaded
in to the program Ingenuity Pathway analysis (IPA 4.0)
from Ingenuity Systems Inc. (Redwood City, CA). The
genes were analyzed by function and those related to
Immune Response were selected. Those genes with a
ratio between 1.5 and -1.25 were excluded.
Cytokine antibody arrays
Panomics TransSignal human antibody arrays 3.0, MA
6160 (Panomics, Inc. Redwood City, CA) were used.
Cell-free supernatants were taken 48 h after infection
and measured without further treatment, according to
the Manufacturer’s instructions. Complete details were
provided in Sharma et al. (2006). Following a thorough
wash of the array membranes, the signals were imaged
with enhanced chemiluminescence (ECL) in a Bio-Rad
Fluor-S Max Multi-imager (Bio-Rad, Hercules, CA),
and signal intensities (average of two identical spots)
were quantified by means of the Bio-Rad “Quantity
One’ software. Results were reproducible on duplicate
membranes.
In several cases, Raybiotech Quantibody arrays were
used to confirm the effects of virus infection on the
secretion of specific cytokines.
Results
CXC chemokines
The chemokines can be conveniently divided into several
sub-groups, depending on their chromosomal location
(many are clustered on chromosome 17, while another
cluster is found on chromosome 4), their detailed
structures and relative sequence homology (most of
them have either the CC or CXC motif), and functional
aspects (chemo-attraction of predominantly neutrophils,
eosinophils, or T-lymphocytes) (Colobran et  al., 2007;
Balestrieri et al., 2008; Vandercappelen et al., 2008).
Figure 1 shows the transcription ratios for four CXC
chemokines located close together on chromosome 4q,
CXCL 9 (Mig), CXCL 10 (IP-10), CXCL11 (I-TAC), and
CXCL13 (BCA; BLC/BCA-1). This group tends to chemo-
attract T-cells, but they are not angiogenic, i.e., do not
promote angiogenesis (Balestrieri et al., 2008), and they
do not possess the amino acid sequence motif ELR.
However, it is evident from Figure 1 that this apparent
similarity is not reflected in their gene responses to the
various treatments. The virus/control ratios (V/C) var-
ied from > 20 for CXCL11 (I-TAC), indicating substantial
up-regulation of that gene, to slight down-regulation of
CXCL13 (BCA). However, E1 and E2, expressed as VE1/V
and VE2/V, respectively, both reversed the viral stimula-
tion, indicating anti-inflammatory activities of the two
Echinacea preparations. The effects of E1 and E2 on con-
trol, uninfected cells (CE1/C and CE2/C in Figure 1) were
quite variable, indicating the variation in individual gene
responses and the differences between the two extracts.
 Echinacea: Anti-inflammatory genes and proteins   503

For comparison, CXCL12 (SDF-1/), which is not part
of this cluster of genes, is also shown in Figure 1.
Corresponding protein data were only available for
CXCL10, and these are shown in Table 1. In spite of the
lack of changes in transcription of this chemokine, there
was a small but significant up-regulation of protein
secretion, which was reversed by both E1 and E2.
Figure. 2 shows the transcription ratios for 5 ELR+
CXC chemokines, most of which are angiogenic, CXCL1
(GRO), CXCL2 (GRO), CXCL3 (GRO), CXCL5
(ENA78), and CXCL8 (also referred to as IL8, interleukin
8). There was relatively little effect of the virus itself, but
in general there was a marked stimulation by both E1
and E2 in uninfected cells, and this was also reflected in
the virus + extract treated cells. However, the responses
of each gene to E1 and E2 were quite different.
Protein data were available for only CXCL1 and 8, and
these are shown in Table 1. For CXCL8 (IL-8), the virus
showed substantial stimulation in protein production,
and this was reversed by the Echinacea extracts. The
25
20
Ratio Normalized Value
CXCL1 protein was not affected by the virus, although
down-regulation by both E1 and E2 occurred. The effects
on uninfected cells were small and did not reflect the
gene transcription changes. Thus, in general, there was
a lack of correlation between gene transcription and the
final products.
CC chemokines
Most of the CC chemokines are clustered on chromosome
17, and many of them chemo-attract neutrophils and/
or eosinophils, in addition to various other functions.
Figure 3 shows the gene array results for CCL2 (MCP-1),
CCL8 (MCP-2), CCL3 (MIP-1), CCL4 (MIP-1), CCL5
(RANTES), CCL11 (eotaxin), CCL17 (TARC) and CCL18
(PARC, MIP-4), expressed as ratios as before. In all cases,
except CCL11, the virus stimulated transcription, espe-
cially CCL 17 and 18, and in most cases this was reversed
by E1 and/or E2. The effects on uninfected cells, however,
25
CXCL 1
Ratio Normalized Value
20 CXCL 2
CXCL 9

CXCL 3
15 CXCL 10 15
10 CXCL 11 CXCL 5
5 CXCL 12 10 CXCL 8
0 CXCL 13
−5 5
−10
−15 0
−20
−5
−25 V/C VE1/V VE2/V CE1/C CE2/C
V/C VE1/V VE2/V CE1/C CE2/C

Figure 1.  Relative gene transcription of CXCL chemokines (ELR-)
9-13. BEAS- 2B cells, in 75 cm 2 flasks, uninfected or infected with
rhinovirus type 14, were treated with Echinacea extract E1 or E2, or
medium only, for 18 h. Total RNA was extracted, converted into cDNA
and hybridized to DNA arrays containing 13,816 genes in duplicate.
Data analysis was carried out as described in Materials and Methods
and the normalized values from three separate experiments were
converted into ratios: V/C = virus/control; VE1/V = virus + E1/virus;
VE2/V = virus + E2/virus; CE1/C = control + E1/control; CE2/C = con-
trol + E2/control.
Figure 2.  CXCL chemokines 1, 2, 3, 5 and 8 (ELR+), relative gene
transcription. BEAS- 2B cells, in 75 cm2 flasks, uninfected or infected
with rhinovirus type 14, were treated with Echinacea extract E1 or
E2, or medium only, for 18 h. Total RNA was extracted, converted
into cDNA and hybridized to DNA arrays containing 13,816 genes
in duplicate. Data analysis was carried out as described in Materials
and Methods and the normalized values from three separate experi-
ments were converted into ratios: V/C = virus/control; VE1/V = virus
+ E1/virus; VE2/V = virus + E2/virus; CE1/C = control + E1/control;
CE2/C = ­control + E2/control.

Table 1.  CXCL chemokines.

Chemokine Principal target cells V/C VE1/V VE2/V CE1/C CE2/C
CXCL1 (GRO) neutrophils 0.64 0.55 2.30 1.07 10.63
(1.01) (0.30) (0.28) (0.48) (0.43)
CXCL2 (GRO) neutrophils 0.85 0.43 3.38 1.22 19.7
CXCL3 (GRO) neutrophils 1.50 2.48 27.4 11.0 45.5
CXCL5 (ENA78) neutrophils 0.5 14.6 0.64 0.48 0.44
CXCL8 (IL-8) Neutrophils,basophils 0.83 5.71 23.76 16.8 35.72
(4.10) (0.06) (0.40) (0.88) (5.6)
CXCL10 (IP-10) Activated T-cells 0.99 1.05 1.04 1.00 1.04
(2.62) (0.50) (0.91) (2.25) (2.90)
Data are expressed as ratios, C = control untreated cells, V = virus infected cells, E1 and E2 = cells treated with Echinacea E1 or E2. The upper
numbers represent gene array data, and the numbers in parentheses represent corresponding ratios from the cytokine antibody results.
504   M. Altamirano-Dimas et al.

were quite variable. Table 2 shows the corresponding
data for the protein products, when these were available.
In general, the protein ratios resembled the correspond-
ing gene changes, although the magnitude of the protein
changes was much less pronounced. Thus, for many
of the CC chemokines transcriptional changes were
reflected in corresponding, but muted, protein changes.
Interleukin clusters
Interleukin 1  and  have long been recognized as pro-
inflammatory cytokines. The genes are part of a large
cluster of related cytokines on chromosome 2, now
classified as IL1 family members 1-11, IL1F1 – IL1F11
20
CCL 2
15
Ratio Normalized Value
(Barksby et al., 2007). Some of these, together with a few
related proteins such as IL1 receptor 1 and caspase  1
(IL1 convertase), were available on the gene arrays.
The data are shown in Table 3. Protein data were also
available for IL1  and , and their receptor antagonist
IL1ra. The virus down regulated the genes for IL1  and
, while IL1 RA was induced. In contrast, both IL1  and
IL1ra proteins were induced. Both E1 and E2 down-reg-
ulated IL1  and  genes in uninfected cells, but these
changes were not reflected in corresponding protein
changes.
The other IL1 family members, as well as caspase 1,
showed variable but small changes in response to virus
infection, and generally a down-regulation in uninfected
cells exposed to Echinacea.
Chromosome 5q31 contains a cluster of cytokines,

CCL 3 IL4, IL5, IL13 and GM-CSF. Their functions, however,

10 CCL 4
5 CCL 5
0 CCL 11
−5 CCL 17
−10 CCL 18
−15
−20
V/C VE1/V VE2/V CE1/C CE2/C
Figure 3.  CCL chemokines 2-5, 11, 17 and 18. BEAS- 2B cells, in 75
cm2 flasks, uninfected or infected with rhinovirus type 14, were treated
with Echinacea extract E1 or E2, or medium only, for 18 h. Total RNA
was extracted, converted into cDNA and hybridized to DNA arrays
containing 13,816 genes in duplicate. Data analysis was carried out as
described in Materials and Methods and the normalized values from
three separate experiments were converted into ratios: V/C = virus/
control; VE1/V = virus + E1/virus; VE2/V = virus + E2/virus; CE1/C =
control + E1/control; CE2/C = control + E2/control.
appear to be unrelated, and this is reflected in their gene
array and protein responses, shown in Table 4. IL4 and
5 showed significant up-regulation in virus-infected
cells, which was generally inhibited by both E1 and
E2, although the corresponding protein changes were
smaller. In contrast, IL13 and GM-CSF were down-
­regulated by the virus, and somewhat up-regulated by
E1 and E2. Thus, there was little relationship between
the members of this cluster at the transcript level, but at
the protein level they were similar.
Other inflammation-related cytokines
A number of other interleukins, and unrelated pro-
teins, have been implicated in various inflammatory
conditions, either as pro-inflammatory mediators or

Table 2.  CCL chemokines, chromosome 17.

Chemokine Principal target cells V/C VE1/V VE2/V CE1/C CE2/C
CCL2 (MCP-1) Monocytes, T-cells, 1.44 0.52 1.03 0.63 4.32
basophils (1.20) (0.41) (0.14) (0.42) (0.43)
CCL8 (MCP-2) Monocytes, T-cells, 7.94 1.02 0.40 5.57 1.72
basophils, eosinophils
CCL3 (MIP-1) same 5.06 2.73 5.0 19.54 5.39
(1.72) (0.47) (0.93) (1.74) (2.82)
CCL4 (MIP-1) same 3.17 1.48 0.51 11.4 22.2
(1.76) (0.34) (1.20) (1.88) (4.0)
CCL5 (RANTES) same 1.45 0.14 0.18 3.70 1.04
(3.10) (0.70) (1.19) (4.24) (4.80)
CCL11 (eotaxin) eosinophils 0.36 3.15 1.16 0.67 0.70
(2.27) (0.63) (1.05) (2.20) (4.70)
CCL17 (TARC) Various T-cells 12.46 0.28 0.06 9.32 9.84
_ _ _ _ _
CCL18 (PARC) Various T-cells 66.3 0.01 0.52 0.02 2.62
(1.40) (0.67) (1.60) (1.80) (2.70)
CCL23 (MPIF) Monocytes T-cells 7.26 0.43 0.59 7.35 0.59
_ _ _ _ _
C = control untreated cells, V = virus infected cells, E1 and E2 = cells treated with Echinacea E1 or E2. The upper numbers represent gene array
data, and the numbers in parentheses represent corresponding ratios from the cytokine antibody results.
 Echinacea: Anti-inflammatory genes and proteins   505

Table 3.  Interleukin-1 family.

IL member Activities V/C VE1/V VE2/V CE1/C CE2/C
IL1F1 (IL-1) Activation of T-cells & 0.40 0.52 0.67 0.46 0.11
macrophages; fever (0.69) (0.55) (0.89) (0.86) (2.43)
IL1F2 (IL-1) same 0.40 1.90 1.11 0.57 0.25
(1.86) (0.40) (1.20) (1.40) (2.93)
IL-1 convertase 0.77 0.31 1.30 0.67 0.55
(caspase-1) _
IL1F3 (IL-1RA) IL-1 receptor 3.90 8.0 0.15 12.6 1.80
antagonist (2.2) (0.31) (0.88) (1.30) (2.80)
IL-1R1 IL-1 receptor 0.50 5.14 2.71 4.38 0.15
_
IL1F4 (IL-18) Induces IFNy; pro- 0.97 0.51 1.10 0.76 1.10
motes Th-1 response _
IL1F7 (IL-1 ) Inhibits IL-18 activity 0.50 – 0.35 0.22 0.59
_
IL1F8 (IL-1 ) Agonist of IL-1 receptor 1.49 0.76 0.47 0.78 0.47
2; upregulates IL-6/8 _
C = control untreated cells, V = virus infected cells, E1 and E2 = cells treated with Echinacea E1 or E2. The upper numbers represent gene array
data, and the numbers in parentheses represent corresponding ratios from the cytokine antibody results.

Table 4.  Cytokine cluster: chromosome 5q31.

Cytokine Activities V/C VE1/V VE2/V CE1/C CE2/C
IL-4 (interleukin-4) Activation of B-cells; differentiation 4.53 0.89 0.25 1.35 0.72
of Th-2 cells (1.20) (1.06) (1.68) (1.95) (1.70)
IL-5 (interleukin-5) Eosinophil growth & differentiation 3.18 1.21 0.01 4.43 2.87
(1.42) (0.92) (1.20) (2.45) (2.60)
IL-13 (interleukin-13) B-cell growth & differentiation; 0.35 1.83 2.84 1.50 0.85
involved in asthma/allergy _
GM-CSF (Granulocyte/­ Growth & differentiation of myelo- 0.19 1.35 5.0 0.44 1.20
macrophage colony- monocyte lineage, particularly (2.24) (1.66) (1.86) (2.65) (6.05)
­stimulating factor) dendritic cells
C = control untreated cells, V = virus infected cells, E1 and E2 = cells treated with Echinacea E1 or E2. The upper numbers represent gene array
data, and the numbers in parentheses represent corresponding ratios from the cytokine antibody results.

as anti-inflammatory regulators (Janeway et  al., 2006).
Gene array data and corresponding protein data are
shown in Table 5. The IL6 protein was substantially
induced by virus, in confirmation of several previous
reports (Message & Johnston., 2004; Sharma et al., 2006;
Edwards et  al., 2007), although gene transcription was
not significantly affected (Table 5, row 1). Both E1 and
E2 inhibited the protein secretion, but transcription was
little affected. IL10 (an anti-inflammatory cytokine) and
IL12 showed relatively small responses in terms of gene
transcription and protein production.
IL16, which has been implicated in asthma and
allergic disorders (Arima et  al., 1999; Deng & Shi,
2006), responded to the virus with marked stimula-
tion at the gene level (Table 5), which was inhibited
strongly by both E1 and E2. We did not have protein
data for this cytokine. IL17 also showed a significant
virus-induction at the protein level, but not at the gene
level (Table 5).
TNF, which has been implicated as a co-inducer of
rhinovirus-induced chemokine expression and airway
inflammation (Newcomb et al., 2007), showed a marked
induction in transcription by the virus itself, but this was
not reflected by protein production (Table 5), in agree-
ment with the report by Gertsch et al. (2004). Echinacea
showed a down-regulation of TNF. In general the pro-
tein changes were considerably less pronounced than
transcription changes, as we observed for some of the
chemokines mentioned above. FAS ligand, a member of
the TNF family, showed smaller effects (Table 5).
The anti-inflammatory TGF and the other cytokines
showed relatively small changes in transcription and
protein production, both in response to the virus and in
response to the Echinacea extracts (Table 5).
A number of other interleukins were also represented
on the gene arrays. These were IL- 2, 3, 7, 9, 11, 13, 14,
15, 19 and 20. Their virus/control ratios varied from 0.15
(IL-19) to 2.13 (IL-3), with relatively small changes in
506   M. Altamirano-Dimas et al.

Table 5.  Cytokines involved in inflammatory conditions.

Cytokine Activities V/C VE1/V VE2/V CE1/C CE2/C
IL-6 Interleukin 6 Growth/ 0.86 1.08 1.04 0.60 0.46
differentiation (5.50) (0.29) (0.44) (2.76) (9.80)
of T & B cells;
fever; acute phase
responses
IL-10 Interleukin 10 Suppresses macro- 1.05 0.56 0.97 0.59 0.93
phage functions (2.30) (0.96) (2.06) (4.50) (5.10)
IL-12 Interleukin 12 Activates NK cells 1.45 0.69 0.68 1.71 1.20
(1.10) (1.20) (1.50) (1.78) (1.50)
IL-16 Interleukin 16 Attracts CD4 T-cells, 8.11 0.18 0.03 7.30 0.13
monocytes & −
eosinophils
IL-17 Interleukin 17 Induces cytokine 0.36 1.60 0.28 0.32 0.14
production in vari- (3.14) (0.61) (0.95) (3.13) (3.80)
ous cells
TNF Tumor ­ Inflammation 44.4 0.62 0.08 18.86 14.44
necrosis factor (1.33) (0.40) (1.16) (1.36) (1.70)
FASL Fas ligand Member of TNF 2.30 1.80 0.15 2.70 1.50
family; apoptosis (2.30) (0.71) (0.70) (2.70) (3.06)
TGF Anti-inflammatory; (1.82) (0.63) (1.08) (1.74) (2.65)
Transforming inhibits cell growth;
growth factor  attracts mast cells
C = control untreated cells, V = virus infected cells, E1 and E2 = cells treated with Echinacea E1 or E2. The upper numbers represent gene array
data, and the numbers in parentheses represent corresponding ratios from the cytokine antibody results.

the other ratios, and protein levels, when available, also
showed minor changes.
Table 6 shows a summary of those cytokines/chem-
okines induced by the virus, at either the gene or protein
level, and their modulation by E1 and E2.
Receptors
A total of eight chemokine receptors and a variety of
interleukin receptors were represented on the gene
arrays. In general, most of the changes in response to
virus or Echinacea were relatively small, and furthermore
there were no apparent correlations between receptors
and the corresponding ligands.
Discussion
The objectives of this study were:
1. to determine how widespread were the effects
of rhinovirus infection on host genes, especially
those genes involved in inflammatory responses
(cytokines and chemokines);
2. to examine the effects of the two distinct Echinacea
extracts on these virus-induced changes, reversal of
virus induction indicating an anti-inflammatory effect;
3. to examine the effects of the Echinacea extracts on
uninfected cells;
4. to determine to what extent the effects shown by
virus and extracts were reflected in changes in
the production of the corresponding proteins, an
absence of correlations indicating the likelihood of
post-­transcriptional controls;
5. to consider how these results can be used to explain
the benefits of Echinacea consumption. The cell
culture-virus model was chosen in order to reflect
the main purpose of Echinacea consumption, which
is to control or alleviate the symptoms of common
colds caused by rhinovirus. Consequently, the prin-
cipal target cells are epithelial cells in the tracheo-
bronchial and nasal tissues.
We and others have reported previously that RV can
bring about substantial changes in gene expression in
human airway epithelial cells (Message & Johnston,
2004; Hudson & Altamirano-Dimas, 2006; Katz et  al.,
2006; Chen et al., 2006; Altamirano-Dimas et al., 2007),
and it is clear from those results and the present data
that many of the gene changes include genes involved
in innate immune responses, particularly chemokines
and other cytokines. Among the collection of cytokines
for which we have presented data here, 14 of them were
significantly induced by the virus, some of them by more
than 5-fold (summarized in Table 6). However, not all
of these resulted in similar changes in the correspond-
ing protein; a total of 13 showed substantial protein
increases, including a few proteins that were induced
in the absence of additional transcription. The latter
 Echinacea: Anti-inflammatory genes and proteins   507

Table 6.  Summary of cytokines showing modulation. array data were not necessarily informative; the protein
Cytokine data were again more convincing. For example, CXCL8
induced by Modulated by transcription was substantially elevated by both E1 and
virus;Gene Modulated by Cytokine induced by E1/E2 E2 in virus-infected and uninfected cells, but the pro-
level E1/E2 or both virus;protein level or both
tein data indicate strong inhibition of the virus-induced
CXCL11 both CXCL8 both
stimulation (Table 1).
CXCL12 E2 CXCL10 both
The IL-1 family of cytokines, which includes IL-18,
CCL8 E2 CCL3 E1
generally showed unremarkable responses to virus and
CCL3 no CCL4 E1
CCL4 E1 CCL5 E1
to Echinacea (Table 3), both at the transcription and pro-
CCL17 both CCL11 E1
tein levels. The other cytokine cluster, containing IL-4,
CCL18 both IL1 E1 5, 13 and GM-CSF, also gave relatively small changes
CCL23 both IL1RA both overall, although the patterns of transcription response
IL1RA E1 GM-CSF no differed widely. The other non-interleukin inflamma-
IL4 E2 IL6 both tory genes also showed few significant responses (Table
IL5 E2 IL10 no 5), except for the anomalous results for TNF, for which
IL16 both IL17 E1 the high stimulations in transcription seen in virus
TNF both FASL both and Echinacea treated cells were not reflected in cor-
FASL E2 responding protein changes, in agreement with Gertsch
et al. (2004).
situation was particularly evident for the well-known Chemokines utilize at least 15 receptors on their
pro-inflammatory cytokines IL-6 and IL-8 (CXCL8), target cells, and there is considerable redundancy
which we and others have reported to be substantially in their usage, such that a specific chemokine could
induced in cells of tracheo-bronchial origin (Message & use several different receptors and a specific receptor
Johnston, 2004; Sharma et al., 2006; Edwards et al., 2007). could bind several different chemokines. Our gene
In both of these cases, transcription was barely affected arrays contained seven CC receptors (CCR 1 – 5, 8, 9)
by virus or by Echinacea, all of the effects shown being and CXCR 1. Their V/C ratios ranged from 0.09 (CCR3)
manifest at the level of protein secretion. However, this to 4.82 (CCR9), but, in any case, a relationship might
should not be surprising in view of the known complex not be significant because these values would reflect
regulatory pathways operating post-transcriptionally transcription and expression of receptors on the source
and post-translationally (Fan et  al., 2005; Proost et  al., epithelial cells, rather than on the target cells (mostly
2006; Gomez-Martin et al., 2008). In fact, IL-8 (CXCL8) leukocytes).
mRNA stability and protein production has been shown Data were also available for a number of interleukin
to be subject to a variety of such controls (Fan et  al., receptors, but again, for the same reasons, most of the
2005). Consequently, in these instances the gene array changes were small and of dubious significance to the
results could give rise to misleading interpretations. overall control of inflammatory conditions.
For this reason it is difficult to draw conclusions about Table 6 summarizes the genes for which virus infec-
the cytokine IL-16, which has been associated with tion resulted in significant induction of either transcrip-
asthma and allergic conditions (Deng & Shi, 2006). If the tion or protein production, and their modulation by the
gene array results presented here reflect protein changes, Echinacea extracts. The two Echinacea preparations did
then this would suggest that not only does RV14 induce not show the same pattern of effects in this virus-cell
this cytokine, but that the Echinacea preparations could system; but this is not surprising in view of the known
prove useful in the treatment or amelioration of such con- significant differences in chemical composition between
ditions. This will have to be confirmed by means of ELISA these two types of preparation, E1 being an aqueous
type tests in the future, since IL-16 was not present on our extract of the above ground parts of the plant, while E2
cytokine antibody arrays. However, the chemokine CCL18 was an ethanol extract of the dried roots (Binns et  al.,
(PARC) showed the highest response of all the cytokines to 2002).
RV infection, and this level was inhibited strongly by both Since we were interested primarily in inflammatory
E1 and E2, yet the corresponding changes in protein levels responses to the virus, then we have not discussed
were relatively small (Table 2). Consequently, we have to cases of viral down-regulation of gene expression,
conclude that the gene array data are not necessarily good although some examples of this were observed.
predictors of protein production, even though for some However, this aspect is relevant to Echinacea consum-
cytokine clusters, such as the group containing CCL-2, 3, ers, based upon the presumption that certain compo-
4, 5 (Table 2), there seemed to be reasonable agreement. nents of Echinacea extracts are capable of neutralizing
In regard to the question of Echinacea as a modula- the virus-induced elevation in secreted chemokines
tor of immune responses induced by the virus, the gene and other cytokines, the latter being responsible for
508   M. Altamirano-Dimas et al.
the myriad symptoms that accompany common colds.
These results also suggest that Echinacea extracts could
be beneficial in treating virus-induced asthmatic and
allergic conditions.
Declaration of interest: The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of the paper.
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