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NEUROENDOCRINOLOGY |
Department of Neurology, Oregon Health Sciences University (A.M., A.A.V., H.O.), Portland, Oregon 97201; L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (A.M.), 53-114 Wroclaw, Poland; Neuroimmunology Research, Veterans Affairs Medical Center (A.M., J.D., A.Z., A.A.V., H.O.), Portland, Oregon 97201; and Department of Molecular Microbiology and Immunology, Oregon Health Sciences University (A.A.V.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Dr. Agata Matejuk, R&D-31, Veterans Affairs Medical Center, 3710 SW U.S. Veterans Hospital Road, Portland, Oregon 97201. E-mail: matejuka{at}ohsu.edu
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Abstract |
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(an important
proinflammatory cytokine in EAE); peptidoglycan recognition proteins
(Pgrp); regulated on activation, normal T cell expressed and secreted
(RANTES); and neural cell adhesion molecule (MCP-1). Up-regulated genes
included cytotoxic T lymphocyte antigen-4 (CTLA-4; known to
inhibit T cell activation), TGFß3, IL-18, and two
interferon-
-induced genes, the chemokines: monocyte chemoattractant
protein-1 (MCP-1) and macrophage inflammatory protein-1ß (MIP-1ß),
vascular cell adhesion molecule (VCAM), and disintegrin metalloprotease
(thought to regulate TNF production). These results implicate a
limited set of known and previously unsuspected E2-sensitive genes that
may be crucial for inhibition of EAE and potentially the human
disease, multiple sclerosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogens are highly pleiotropic, affecting a wide variety of cells and
tissues. According to the current view, estrogens mediate their
activities through transcription-regulating intracellular estrogen
receptors (iER). These proteins contain several domains for estrogen
binding, nuclear localization, dimerization, DNA binding, and
trans-activation that impart to iERs the ability to activate
or repress specific estrogen-responsive genes. The influence of E2 was
demonstrated on the production of several cytokines, such as IL-1
(18), IL-6 (19) TNF (20), and
interferon- (IFN) (21). The cytokine secretion
pattern of human CD4+ T cell clones was strongly
influenced by high concentrations of E2 (22, 23).
Recently, we demonstrated a significant decrease in the frequency of
TNF-producing cells in the CNS and the periphery in mice treated
with estrogen (24).
Two different iERs have been characterized, iER and the newly
discovered iERß, that may have overlapping or distinct functions. To
account for the myriad of distinct tissue-specific effects, it has been
suggested that different iER modulators may induce conformational
changes in the receptor that result in a specific biological activity.
Several studies implicate a functional ER found on human blood
mononuclear cells, splenic and thymic cells (25), as well
as human leukocyte antigen-DR-negative human peripheral and thymic T
cells (26) and the CD8 subset of human T cells (27, 28). Our recent study demonstrated ER message in purified
murine CD4+ T cells (29).
Additionally, a novel E2 pathway through plasma membrane receptors was
demonstrated in CD4+ as well as
CD8+ T cells (30).
In this study we investigated the effects of changes in the expression of more than 12,000 genes caused by hormonal treatment to understand the complexity of genetic events leading to protection from autoimmune diseases like EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of active EAE
BV8S2/AV4 double Tg female mice were immunized with 400 µg
myelin basic protein (MBP)-NAc1-11 (Ac-ASQKRPSQRSK) in complete
Freund’s adjuvant (CFA) containing 200 µg
Mycobacterium tuberculosis by sc injection over four sites
on the flank on d 0. Mice were assessed daily for clinical signs of EAE
according to the following scale: 0 = no signs; 1 = limp
tail; 2 = moderate hind limb weakness (waddling gait); 3 =
moderately severe hind limb weakness; 4 = severe hind limb
weakness; 5 = paraplegia; and 6 = quadriplegia, moribund
condition. At the peak/acute phase of EAE (maximum severity of clinical
signs, 14th day after immunization with encephalitogenic peptide),
representative mice from EAE and estrogen-treated groups and two
control groups (naive and CFA) were killed, and spleens were removed
surgically and passed through a wire mesh screen to obtain a single
cell suspension. Frozen splenocyte cells were subsequently subjected to
total RNA extraction using the STAT-60 reagent (Tel-Test,
Friendswood, TX).
Estrogen treatment
For estrogen hormone therapy 3-mm pellets containing 2.5 mg E2
(1500–2000 pg/ml serum, 20–50% of pregnancy levels; Innovative Research of America, Sarasota, FL) were implanted sc on the back
7 d before induction of EAE. EAE mice were sham operated and were
implanted with a saline pellet (which did not affect the course of EAE;
our unpublished data) or no pellet. The estrogen pellets provide
continuous controlled release of a constant level of hormone over a
period of 60 d. Serum concentrations of estrogen monitored before
and during the course of EAE in representative control and implanted
mice consistently fell within the expected ranges as measured by RIA
method.
GeneChip array assay
Sample labeling. Isolated total RNA was labeled as described
in the Affymetrix (Santa Clara, CA) GeneChip Expression Analysis
Technical Manual (revision 3). The labeling is performed in two steps.
In the first step, mRNA is converted to double stranded cDNA using
SuperScript Reverse Transcriptase (Life Technologies, Inc., Carlsbad, CA) and an oligo(deoxythymidine) primer linked
to a T7 RNA polymerase-binding site sequence. In the second step, cDNA
is converted to labeled cRNA (the target) using T7 RNA polymerase in
the presence of biotinylated UTP and CTP (Enzo Diagnostics,
Farmingdale, NY). This step also provides a linear amplification of the
labeled material. After removal of free nucleotides, target yield is
measured by UV260 absorbance.
Target quality assessment. The labeled target is fragmented at 95 C in the presence of high magnesium concentration. The fragmented material is combined with control oligomer (used for grid alignment during image processing) and control cRNAs for BioB, BioC, BioD, and Cre (Affymetrix) in hybridization buffer. Four micrograms of target is hybridized with a Test Array (Affymetrix) overnight and then washed and stained on the Fluidics Station. The Test Array contains probe sets for all Affymetrix controls (e.g. BioB, BioC, BioD, Cre, and murine actin and glyceraldehyde-3-phosphate dehydrogenase). After image processing and absolute analysis of the array pattern with MAS (see Data analysis section), four values were examined: background, noise, average difference, and ratio of average difference values for probe sets representing the 5'- and 3'- ends of actin and glyceraldehyde-3-phosphate dehydrogenase transcripts. Cut-off values are determined within the project sample set, and all targets that do not meet these thresholds will be remade whenever possible or discarded from further analysis.
GeneChip genome array hybridization. Fragmented targets are combined with control oligomer and control cRNAs in hybridization buffer. Depending on the sensitivity required and the yields of target achieved, 10 -20 µg labeled cRNA were used for genome array hybridization. Each target was hybridized to MG_U47A using protocols described in the Affymetrix Expression Analysis Technical Manual. Was performed overnight, followed by washing, staining, signal amplification with biotinylated antistreptavidin antibody, and a final staining step. The distribution of fluorescent material on the array was measured using the laser scanner, and the resultant image was processed with MAS software.
Data analysis
The Bioinformatics and Biostatistics component of the Microarray
Core performed data preparation, data analysis, and data integration.
Data preparation involves image processing, evaluation of data quality,
normalization, transformation, and filtering so that data are ready for
further analysis. Data analysis involves two- and three-dimensional
data visualization to more sophisticated multivariate analysis, such as
cluster analysis, principal component analysis, and multidimensional
scaling. Data integration involves the development and maintenance of
integrated databases consisting of gene expression data, sample and
experiment information, analysis results, and biological information.
This step includes identification of gene functions through access to
existing public databases.
Data presentation
All data presented in Tables 1
and 2 show the mean fold change (decrease or
increase) in gene expression with SD from two independent
experiments in estrogen-treated mice compared with EAE mice with severe
clinical signs of disease. The variation among all 12,488 genes between
the 2 experimental evaluations was less than 0.03 in mice with EAE and
less than 0.01 for estrogen-treated mice. The mean fold minimum change
chosen for presentation in the category estrogen over EAE was 2.4, and
the SD was no more than 30%, resulting in a significant
change (P < 0.05), as determined by a t
test run on an Excel PC program. All genes presented in Tables 1 and 2
were significantly changed by estrogen treatment. Other transcriptional
changes in gene of interest (CFA over naive, EAE over CFA, EAE over
naive) are also presented in Tables 1 and 2. The variation between the
two evaluations was less than 0.02 in naive mice and less than 0.02 for
CFA-immunized mice.
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, monocyte
chemoattractant protein-1 (MCP-1), and T cell activation antigen-3; and
C chemokine: lymphotactin, and the following cytokine
transcripts: IL-4, IL-10, TNFß, Lt-ß, TNF, IL-6, IFN, IFNß,
TGFß1, TGFß2, TGFß3, and macrophage migration inhibitory
factor. The chemokine receptor set detected the following
transcripts: chemokine receptor 1 (CCR1), CCR1b, CCR2, CCR3, CCR4, and
CCR5. The sample loading was normalized by the housekeeping gene, L32,
included in each template set. RPA analysis was performed on 5 µg
total RNA hybridized with probes labeled with
[-32P]UTP. After digestion of
single-stranded RNA, the RNA pellet was solubilized and resolved on a
5% sequencing gel. Controls included the probe set hybridized to tRNA
only; appropriate control RNA, which serves as integrity control for
the RNA sample; and yeast tRNA as a background control. For
quantification, gels were exposed by phosphorimaging (Bio-Rad Laboratories, Inc., Hercules, CA), and radioactivity in
individual bands (after background subtraction) in comparison with L32
was assessed with Quantity One software (Bio-Rad Laboratories, Inc.). |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) as well as from control groups in two independent
experiments (Fig. 1). The pattern of gene
expression varied by 1–3% between the first and second experiments
for each respective control and experimental group.
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Females implanted with 2.5-mg pellets of E2 did not develop any sign of
disease 14 d after immunization with MBP-NAc1–11 peptide/CFA. In
contrast, control mice not receiving E2 displayed severe hind limb
weakness and paraplegia. Serum E2 levels in mice implanted with
estrogen ranged from 1000–2000 pg/ml.
Genes with increased expression
Table 1 presents genes categorized into five groups, exhibiting
from 2.5- to 9.8-fold mean increase in expression, with an
SD of no more than 30% observed in female mice treated
with E2 (protected from EAE) compared with untreated mice females with
severe EAE. Among cytokine genes, the highest mean fold change of +9.8
was observed for IFN-induced 15-kDa protein (ISG15). Three other
cytokine genes were also up-regulated, including IFN-induced
protein with tetratricopeptide repeats 1 (GARG-16), IL-18, mouse mast
cell growth factor, and TGFß3. Among up-regulated chemokine genes,
the greatest change (+6.0) was observed for MCP-1, described as small
inducible cytokine A2, but also known as macrophage chemoattractant
protein-1, followed by MIP-1ß. Additionally, expression of the CCR3
gene, often found on Th2 and mast cells, was up-regulated 2.8-fold.
Besides cytokines, chemokines, and their receptors, we were interested in whether estrogen treatment was able to induce changes in transcriptional properties in adhesion molecule and activation marker genes. mRNA for disintegrin metalloprotease (decysin) showed an 8.4-fold increase, and other adhesion molecules, including guanine nucleotide exchange factor and integrin-binding protein homolog GRP1, and vascular cell adhesion molecule 1 (VCAM-1), were also up-regulated. Up-regulated activation marker genes included 4F2/CD98 light chain, Ig superfamily member cytotoxic T lymphocyte antigen-4 (CTLA-4), and CD14 antigen.
Genes with decreased expression
Overall, 187 genes displayed a 2.4-fold or greater decrease with
an SD of 30% or less after estrogen treatment. Table 2
presents a list of two cytokine-related, one chemokine, and one
adhesion molecule genes that were affected by estrogen therapy. The
greatest decrease was found in the TNF gene, which showed a more
than 10-fold decrease in expression. The other cytokine-related gene,
Pgrp (TNF superfamily 3-like), was moderately down-regulated. Also
down-regulated by E2 treatment was the chemokine RANTES and neural cell
adhesion molecule (NCAM).
Changes in estrogen and other hormone-related genes
Surprisingly, we were unable to see any transcriptional changes of
more than 2.5-fold in known estrogen-related genes in E2-treated
EAE-protected mice vs. control mice with EAE. The only
noteworthy changes included a 2-fold decrease in ERß and a 1.6-fold
increase in ER-related protein.
Changes in other genes in E2-treated vs. untreated mice with severe
EAE
In addition to immune response-related genes, the Affymetrix
analysis detected a total of 260 up-regulated genes and a total of 187
down-regulated genes with a mean fold change of 2.4 or more and an
SD less than 30%. These changes are available in an EXCEL
file, estrogen over EAE 2.4, from the corresponding author.
Changes in gene expression in CFA-treated animals over naive
animals
Immunization with CFA alone did not cause EAE, and induced
down-regulation of 41 genes and up-regulation of 112 genes compared
with naive untreated mice. A listing of these genes can be found in the
EXCEL file CFA over naive 2.4 (available from the corresponding
author). Changes in the expression of genes of interest in the category
CFA over naive are presented in Table 1 for up-regulated genes and in
Table 2 for down-regulated genes.
Changes in gene expression in EAE vs. CFA-treated animals
Immunization with MBP-Ac1–11 encephalitogenic peptide emulsified
in CFA induced down-regulation of 206 genes and up-regulation of 138
genes vs. those in animals immunized with CFA only. A
listing of these genes can be found in the EXCEL file EAE over CFA 2.4
(available from the corresponding author). Changes in the expression of
genes of interest in the category EAE over CFA are presented in Table 1
for up-regulated genes and in Table 2 for down-regulated genes.
Changes in gene expression in EAE vs. naive treated
animals
Immunization with MBP-NAc1–11 peptide induced down-regulation of
152 genes and up-regulation of 251 genes compared with naive mice. A
listing of these genes can be found in the EXCEL file EAE over naive
2.4 (available from the corresponding author). Changes in the
expression of genes of interest in the category EAE over naive are
presented in Table 1 for up-regulated genes and in Table 2 for
down-regulated genes.
Changes in genes found by the RPA technique
Using the RPA technique, a specific and sensitive method that
allows detection and quantification of mRNA species, we were able to
verify selected portions of the Affymetrix data. Figure 2 shows the
mean fold decrease or increase in mRNAs in estrogen-treated compared
with untreated mice as detected by RPA. Although the fold numbers are
not exactly the same as those obtained by the Affymetrix technique, the
increase or decrease in mRNA expression after estrogen treatment was
observed in the same genes as those found using Affymetrix. RANTES and
TNF mRNA levels were down-regulated about 2-fold in E2-treated mice.
Similar to Affymetrix results, up-regulation of mRNA levels was
observed for MCP-1 (2-fold), TGFß3 (6-fold), and CCR3 (3.7-fold).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study revealed some interesting findings in the expression of genes for several cytokines, chemokines, and adhesion molecules as well as their receptors after treatment with E2. One of the major outcomes from our study is that estrogen treatment is restricted. Less than 10% of the genes tested were affected by hormone therapy. Nearly 200 genes were down-regulated (2.4-fold), and more than 200 genes were up-regulated (2.4-fold) during estrogen therapy. For the majority of these genes, the direct relation to EAE disease and/or estrogen therapy is still unknown, and changes in mRNA and protein expression still need confirmation using other techniques.
E2 had profound effects on the expression of a set of only 18 immune
response-related genes detected in splenocytes from E2-treated mice
that were fully protected from EAE vs. mice with severe EAE.
As is shown in Table 2, the cytokine most inhibited by E2 treatment was
TNF (decreased 10.4-fold). Inhibition of TNF, Pgrp [another TNF
superfamily member involved in innate immune responses
(33)], RANTES [known to be increased in the CNS in
MS and EAE (34, 35, 36)], and NCAM [increased in
cerebrospinal fluid from patients with active MS (37, 38)] might well be directly related to E2-dependent enhancement
of CTLA-4 [a costimulatory molecule known to down-regulate T cell
activation (39)], TGFß3 [known to have potent
antiinflammatory effects and to be E2 sensitive (40)],
GRP-1 [binds phosphoinositides involved in signal transduction
(41)], and disintegrin metalloprotease [found on mature
dendritic cells and thought to regulate TNF production
(42)]. The possible role of other E2-stimulated genes in
splenocytes is more speculative. E2 clearly up-regulated IL-18
(IFN-inducing protein) (43, 44) and two IFN-induced
proteins, ISG15-IFN-stimulated gene (45) and GARG-16
(46), suggesting that these factors could be associated
with the previously described EAE-protective effects of IFN.
Moreover, E2 up-regulated the chemokines MCP-1 [increased in mice
orally tolerized against encephalitogenic peptides (47, 48)], MIP-1ß [increased in CNS during EAE and MS (35, 49), but also increased in lymph nodes of mice protected from
EAE by T cell antigen receptor-directed therapy (36)],
and VCAM (major component mediating leukocyte-endothelial
adhesion; induction by TNF and IL-1ß is E2 sensitive (50, 51)], as well as mast cell growth factor [promotes growth of
mast cells involved in EAE (52)] and CCR3 [Th2 and mast
cell chemokine receptor found to be up-regulated in brains of T
cell antigen receptor-protected mice (36, 53, 54)].
Up-regulation of chemokines and adhesion molecules within the spleen
may attract and retain inflammatory cells and thus reduce their
migration into the CNS. Least understood in E2-mediated protection
against EAE is up-regulation of the CD98 light chain [involved as a
heterodimer in cellular activation, proliferation, and adhesion
(55, 56)] and CD14 [known E2-sensitive receptor for LPS
on Kuppfer cells (57)]. Many other genes were affected to
a lesser degree.
RPA analysis of splenocyte gene expression confirmed E2-associated
down-regulation of TNF and RANTES and up-regulation of MCP-1,
TGFß3, and CCR3. E2 inhibition of intracellular TNF protein
expression has been clearly documented in our previous study
(24). However, conclusions regarding other E2-sensitive
genes remain tentative until changes in protein expression can be
verified individually. Overall, these data provide strong confirmation
of the inhibitory effects of E2 on TNF in spleen and identify a
limited set of known and previously unsuspected candidate genes that
may contribute to E2 regulation of EAE. Many of these genes may be
involved in apoptotic mechanisms related to the effects of TNF. The
mechanism by which E2 inhibits EAE has been only partially identified
as inhibiting TNF and migration of inflammatory cells into the CNS.
TNF is an important effector molecule in EAE, produced by
inflammatory macrophages and T cells, but its detrimental effects
apparently do not include overt apoptosis of CNS cells (such as
oligodendroglial cells) in vivo, at least during the acute
stages of EAE evaluated here. It is interesting that E2 prevents
apoptosis in other organs, including ovarian tissues, and it is
conceivable that this activity in some way could contribute to the
inhibition of demyelination and EAE.
In conclusion, E2 treatment affected about 10% of the genes tested,
but only 18 cytokine, chemokine/receptor, adhesion molecule, or
activation genes were up- or down-regulated more than 2.4-fold by E2
treatment. Down-regulated genes included TNF (an important
proinflammatory cytokine in EAE), Pgrp, RANTES, and NCAM. Up-regulated
genes included CTLA-4 (known to inhibit T cell activation), TGFß3,
IL-18, and two IFN-induced genes, the chemokines MCP-1 and MIP-1ß,
VCAM-1, and disintegrin metalloprotease (thought to regulate TNF
production). These results implicate a limited set of known and
previously unsuspected E2-sensitive genes that may be crucial for
inhibition of EAE and potentially the human disease, MS. The
constellation of up-regulated and down-regulated genes and the
understanding of their transcriptional changes can lead to a better
understanding of the complexity and the mechanism of action of a
variety of drugs, including hormone treatment.
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Footnotes |
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1 Postdoctoral fellow of the National Multiple Sclerosis Society. 
Abbreviations: CCR, Chemokine receptor; CFA, complete Freund’s
adjuvant; CNS, central nervous system; CTLA, cytotoxic T
lymphocyte antigen; EAE, experimental autoimmune
encephalomyelitis; GRP, guanyl nucleotide-releasing protein; iER,
intracellular ER; IFN, interferon-; MCP-1, monocyte
chemoattractant protein-1; MBP, myelin basic protein; MIP,
macrophage inflammatory protein; MS, multiple sclerosis; NCAM, neural
cell adhesion molecule; Pgrp, peptidoglycan recognition proteins;
RANTES, regulated on activation, normal T cell expressed and secreted;
RPA, ribonuclease protection assay; VCAM, vascular cell adhesion
molecule.
Received July 11, 2001.
Accepted for publication September 10, 2001.
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References |
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production by T cells. Nature 378:88–91[CrossRef][Medline]
by NK cells. J Immunol 165:3099–3104This article has been cited by other articles:
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