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,§
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Departments of
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Neurology and
Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201;
L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; and
§
Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-1ß, and MIP-2; whereas brain had less
infiltration and a lower expression of a different pattern of
chemokines and receptors. In TCR-protected mice, there was a decrease
in the number of inflammatory cells in both SC and brain. In SC, the
reduced cellular infiltrate afforded by TCR vaccination was
commensurate with profoundly reduced expression of chemokines and their
cognate chemokine receptors. In brain, however, TCR vaccination did not
produce significant changes in chemokine expression but resulted in an
increased expression of CCR3 and CCR4 usually associated with Th2
cells. In contrast to CNS, lymph nodes of protected mice had a
significant increase in expression of MIP-2 and MIP-1ß but no change
in expression of chemokine receptors. These results demonstrate that
TCR vaccination results in selective reduction of inflammatory
chemokines and chemokine receptors in SC, the target organ most
affected during EAE. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies conducted in our laboratory on transgenic (Tg) mice
expressing BV8S2 specific for the myelin basic protein
(MBP)-NAc1–11 peptide revealed that protection
against EAE can be induced by TCR BV8S2-specific regulatory T cells
through a nondeletional cytokine-driven suppressive mechanism
(11). In these mice, essentially all of the T cells
express the Tg BV8S2 chain, paired with a naturally rearranged TCR
-chain. Vaccination with heterologous rat BV8S2 protein resulted in
the maturation of BV8S2-specific regulatory T cells, inhibition of
MBP-specific Th1 cells, and protection against EAE. Protection was
associated with a decrease in IFN-
production by T cells specific
for the encephalitogenic MBP-NAc1–11 determinant
and increased production of IL-10 by BV8S2-reactive T cells in lymph
nodes (LN), and reduced cellular infiltration into the CNS. However,
the effects of TCR vaccination on expression of chemokines and their
receptors and the mechanisms that control lymphocyte trafficking from
LN to CNS have not yet been evaluated.
Chemokines most likely participate in the pathogenesis of autoimmune
disorders. Elevated expression of monocyte chemoattractant protein-1
(MCP-1), macrophage-inflammatory protein-1 (MIP-1),
macrophage-inflammatory protein-1ß (MIP-1ß), RANTES, and
IFN-inducible 10-kDa protein (IP-10) was observed in the CNS of
patients with multiple sclerosis (MS), and animals with EAE
(12, 13, 14, 15, 16, 17), emanating from infiltrating mononuclear cells as
well as resident CNS cells including microglia, astrocytes, and
endothelial cells (13, 17). Abs to MIP-1 and MCP-1
ameliorated disease (14, 18, 19). Much less is known about
expression of chemokine receptors in MS and EAE. Increased levels of
CCR2 and CCR5 were found in spinal cords (SC) of rats displaying
clinical signs of EAE (20). In MS brain, infiltrating T
cells were CCR5 and CXCR3 positive, in accordance with an increased
expression of their ligands, MIP-1 and RANTES, and, respectively,
IP-10 in demyelinating lesions (21, 22).
In this study, we have characterized the chemokine and chemokine
receptor profile in CNS (SC and brain) and peripheral LN in TCR BV8S2
Tg mice during the acute stage of EAE 15 days after immunization with
encephalitogenic MBP-NAc1–11 peptide in CFA with
or without prior vaccination with the BV8S2 protein. We evaluated the
level of mRNA expression of several members of the CC subfamily:
eotaxin, RANTES, MIP-1, MIP-1ß, and MCP-1; the CXC subgroup: MIP-2
and IP-10; and one member of C subfamily: lymphotactin (Ltn), as well
as the chemokine receptors CCR1, CCR1b, CCR2, CCR3, CCR4, and CCR5. We
found strong up-regulation in the expression of inflammatory chemokines
RANTES, IP-10, MCP-10, MIP-1, MIP-1ß, and MIP-2 and chemokine
receptors CCR1, CCR2, and CCR5 in the SC of animals displaying clinical
signs of EAE that were profoundly reduced in EAE-protected mice
vaccinated with the BV8S2 peptide. Interestingly, brain chemokine and
chemokine receptor expression was different from that found in SC. In
contrast to CNS, LN of protected mice had a significant increase of
MIP-2 and MIP-1ß chemokines but no changes in expression of chemokine
receptors. These findings demonstrate that TCR-based immunoregulation
results in selective reduction of chemokines and chemokine receptors in
the target organ most affected during EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total RNA was extracted from frozen SC and brain tissues, and
lymphocytes were isolated from LN using the STAT-60 reagent (Tel-Test,
Friendswood, TX). Chemokine expression was determined by using the
RiboQuant RPA kit (PharMingen, San Diego, CA) according to the
manufacturer’s instructions. A multiprobe set detected the following
chemokine transcripts: CXC chemokines: MIP-2 and IP-10; C-C chemokines:
RANTES, eotaxin, MIP-1ß, MIP-1, MCP-1, and T cell activation Ag
(TCA-3); and C chemokine: Ltn. The chemokine receptor set detected the
following transcripts: 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 ssRNA, 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, Hercules, CA),and radioactivity
in individual bands (after background subtraction) in comparison with
L32 was assessed with Quantity One software (Bio-Rad).
Mice
Tg mice bearing the rearranged BV8S2 gene on the B10.PL background (23) were kindly provided by Dr. Joan Goverman (Seattle, WA). Male Tg mice were bred with B10.PL females, and 4-wk-old offspring were screened for BV8S2 TCR expression by FACS analysis. The colony was housed and cared for in the Animal Resource Facility at the Portland VA Medical Center according to institutional guidelines. Mice were used at 8–12 wk of age. Ags: N-acetylated MBP1–11 peptide (Ac-ASQKRPSQRSK) was synthesized by solid phase techniques and was purified by HPLC at the Beckman Institute, Stanford University (Stanford, CA). GST-BV8S2 proteins were expressed and purified as described previously (24). The GST-BV8S2 fusion protein contains the complete BV, BD, and BJ regions and the first 19 residues of the BC region from the TCR of an encephalitogenic rat T cell clone fused to the C-terminal end of GST.
Chemokine ELISA
SC were homogenized in PBS with a tissue homogenizer. Debris was
removed by centrifugation, and the aqueous extract was collected at
-70°C until total protein determination and ELISA assessment of
chemokines. Secretion of RANTES, MIP-1, and MCP-1 proteins was
assessed using commercially available immunoassay kits purchased from
R&D Systems (Minneapolis, MN) according to the manufacturer’s
instructions. ELISA was normalized by total protein determination and
expressed as picograms per milligram of tissue.
Induction of active EAE and protection with BV8S2 protein
BV8S2 Tg males were immunized with 400 µg
MBP-NAc1–11/CFA containing 200 µg
Mycobacterium tuberculosis by s.c. injection over four sites
on the flank on day 0. For protection experiments, mice were injected
with 12.5 µg of recombinant rat BV8S2 protein/IFA i.p. on days -7
and +3 relative to injection of the MBP-NAc1–11
according to the protocol developed by Kumar and Sercarz
(25). 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; 6 = quadriplegia, moribund
condition. Onset was defined as the first day of clinical signs, and
peak (acute phase of EAE) as maximum severity of clinical signs (14–16
days after immunization with encephalitogenic peptide). The cumulative
disease index was determined for each mouse by summing the daily
clinical scores (Table I
). Representative mice from control and
protected groups were sacrificed and SC, brains and LN were taken for
gross weight, quantification of infiltrating cells, and analysis of
chemokines and receptors. SC were isolated by insufflation and brains
were removed surgically. Mononuclear cells were isolated over a Percoll
step gradient and counted as previously described (26). LN
were removed surgically and passed through a wire mesh screen to obtain
a single-cell suspension. SC and brain tissues as well as lymphocytes
from LN were frozen at -70°C and subsequently thawed and evaluated
for expression of chemokines and chemokine receptors by the RNase
protection assay. Additionally, SC were evaluated for chemokine
proteins by ELISA as described above.
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Comparisons between two groups were analyzed using Student’s t test. The accepted level of significance was p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To boost the naturally induced response to BV8S2 determinants,
BV8S2 Tg mice were vaccinated with heterologous (rat) BV8S2 protein on
days -7 and +3 relative to induction of EAE with
MBP-NAc1–11. To sustain responses to BV8S2
protein, protected mice were boosted on day +10 with BV8S2 protein.
Protected mice had a significantly lower incidence of EAE (10% vs
82%, result of three different experiments, p <
0.0001), and as a group, a significantly lower peak disease score and
cumulative disease index than control mice immunized with
MBP-NAc1–11/CFA but not treated with BV8S2
protein (Table I
). Protection against
clinical EAE was reflected by a marked reduction of total inflammatory
mononuclear cells and T cells, which comprised 50–60% of the total
cell infiltrate, in SC and brain (Table II).
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To optimize detection of clinically important changes, chemokine
expression was compared in EAE protected vs control mice during the
acute phase of disease that occurs 14–16 days after immunization with
NAc1–11 peptide/CFA. A quantitative method, the
RPA, was used to examine RNA synthesis for the following chemokines:
Ltn (C subfamily); RANTES, eotaxin, MIP-1ß, MIP-1, MCP-1, and
TCA-3 (C-C subfamily); and MIP-2 and IP-10 (CXC subfamily). The tissue
sample for RPA was prepared by homogenization of whole SC and total
cellular RNA was extracted using mRNA isolation reagent. As shown in
Fig. 1 and quantified in Fig. 2, transcripts for RANTES, MIP-1ß,
MIP-1, MIP-2, IP-10, and MCP-1, but not Ltn, eotaxin, and TCA-3,
were detected in SC from both control and protected mice during the
acute phase of EAE. Message for Ltn and eotaxin was slightly detectable
only after overloading the gel. Paraplegic mice (control group) had
abundant RNA expression of IP-10, RANTES, and MCP-1, with lesser mRNA
levels of MIP-1ß, MIP-1, and MIP-2 as anticipated (Fig. 2). In
contrast, TCR-protected mice had profoundly lower levels of mRNA
expression of all chemokines tested (RANTES, p = 0.02;
MIP-1ß, p = 0.045; MIP-1; p =
0.014; MIP-2, p = 0.007; IP-10, p =
0.002; MCP-1 p = 0.03; Fig. 2).
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Chemokine protein expression for RANTES, MIP-1, and MCP-1 was
determined using commercially available immunoassay kits. ELISA
analysis of RANTES and MCP-1 confirmed a striking decrease in these
chemokines in the SC of EAE-protected mice compared with controls with
severe EAE (RANTES, p < 0.001; MCP-1,
p < 0.014; MIP-1, p < 0.003; Fig. 3). Results were normalized to the
protein content of the total SC homogenate.
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The RPA technique was also adopted to determine the expression of
chemokine receptor genes. As shown in Fig. 4, message was detectable only for CCR1,
CCR2, and CCR5 in SC samples from both EAE-protected and control mice,
whereas message for CCR1b, CCR3, and CCR4 was not detectable by RPA in
any of the extracts. Elevated levels of mRNA for CCR1, CCR2, and CCR5
were observed in SC of mice with severe EAE, but up to 8-fold lower
mRNA levels were found in SC of EAE-protected mice
(p < 0.005 for CCR1 and CCR5;
p < 0.05 for CCR2; Fig. 4).
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Similar measurements were conducted in brain tissue at the peak of
disease from paralyzed untreated controls and EAE-protected mice. As in
SC samples, there was elevated expression of RANTES, IP-10, and MCP-1,
but no detectable, or only slightly detectable (on overloading of gel),
expression of MIP-1 and MIP-2 in control mice with EAE (Fig. 5). However, in brain, there were no
statistically significant differences in expression of any chemokine in
protected vs control mice, with the transcript levels for RANTES being
nominally higher in brains of sick mice, that for IP-10 being the same
and that for MCP-1 being slightly higher in brains of protected
animals.
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The pattern of expression of chemokine receptors was different in
brain vs SC tissue. In at least three separate experiments of brain
tissue, a relatively low level of message 10- to 20-fold less than that
in SC was detected for all of the chemokine receptors tested, including
CCR1, CCR1b, CCR2, CCR3, CCR4, and CCR5. However, it is noteworthy that
brain tissue from mice protected from EAE had a significantly lower
expression of a chemokine receptor associated with Th1 cells (CCR1) but
a significantly higher expression of receptors associated with Th2
cells (CCR3 and CCR4) (Fig. 6).
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As determined by RPA, peripheral LN showed a different pattern of
chemokine expression than that observed in CNS (Fig. 7). In contrast to both brain and SC
tissue, no message could be detected for MCP-1, although message for
Ltn was present as expected. LN cells expressed easily detectable
levels of message for all other chemokines except eotaxin, with RANTES
being predominant. However, unlike SC tissue, LN cells from protected
mice had slightly increased levels of chemokine message compared with
control mice with EAE, with significantly elevated expression of
MIP-1ß and MIP-2 (p = 0.006 and
p = 0.004, respectively; Fig. 7).
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RPA analysis indicated that peripheral LN expressed mRNA for CCR1,
CCR2, CCR4, and CCR5 but not CCR1b and CCR3 (Fig. 8). The predominant chemokine receptor in
LN was CCR2, but no differences were observed in protected vs control
mice in expression of any of the chemokine receptors (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MCP-1, and TCA-3), and two
C-X-C-chemokines (IP-10 and MIP-2) were quantified in parallel with CC
chemokine receptors (CCR1, CCR1b, CCR2, CCR3, CCR4, and CCR5) in CNS
and peripheral LN of Tg mice with EAE vs mice protected from EAE by
vaccination with TCR protein.
The results presented above provide a number of unique insights. First,
in control mice paralyzed with EAE, expression of the chemokines
RANTES, IP-10, and MCP-1 occurred predominantly in the SC at much
higher levels than in brain or LN. MIP-1ß, MIP-1, or MIP-2 were
also expressed in SC but were not detectable in brain. Both SC and
brain had considerably elevated numbers of inflammatory cells and T
cells per mg of tissue (Table II), but cellular infiltration was about
4 times greater in SC, most likely contributing to the ascending
paralysis that is typical of rodent EAE. Inflammation in the brain also
may have had distinct neurological consequences that were not
quantifiable.
The enhanced 3.5-fold expression of some chemokines in SC (e.g.,
RANTES) would thus appear to be correlated with the 4-fold increase in
mononuclear cell infiltration. However, the elevated degree of
expression in SC vs brain of IP-10 (>10-fold), MCP-1 (>20-fold), and
MIP-1ß, MIP-1, and MIP-2 that were only detectable in SC was
clearly not commensurate with the 4-fold elevation in SC inflammatory
cells. Moreover, LN cells had greatly reduced or absent levels of
RANTES, IP-10, and MCP-1 relative to SC. Taken together, these findings
indicate that elevated production of inflammatory chemokines was tissue
associated and not simply related to the number of mononuclear cells
present. Selective and elevated expression of CCR1, CCR2, and CCR5 in
SC lends support to the concept that co-localization of these chemokine
receptors shared primarily by effector Th1 cells and monocytes could
promote a delayed type hypersensitivity-type reaction (30)
that is typical for CNS lesions in EAE. Differences in expression of
chemokines and receptors in brain tissue may reflect qualitative and
quantitative differences in infiltrating and resident cell types, a
matter for further investigation.
Our study was designed specifically to determine whether vaccination
with TCR BV8S2 protein would alter expression of chemokines and their
receptors. In mice protected from EAE by TCR vaccination, there was a
marked (70–80%) decrease in the number of total inflammatory cells
and T cells that infiltrated into both SC and brain tissues. In SC, the
reduced cellular infiltrate afforded by TCR vaccination was
commensurate with profoundly reduced expression of inflammatory
chemokines and their cognate chemokine receptors CCR1, CCR2, and CCR5.
In brain, however, TCR vaccination did not produce significant changes
in the expression of RANTES, IP-10, or MCP-1. TCR vaccination reduced
expression of CCR1 in the brain of protected mice, with no effect on
CCR2 or CCR5, but increased message for chemokine receptors CCR3 and
CCR4 that have been associated with Th2 responses (e.g., TCR-reactive T
cells). In LN, TCR-induced protection from EAE was reflected by
increased expression of MIP-1ß and MIP-2, with no change in
expression of RANTES, IP-10, MIP-1, Ltn, or chemokine receptors.
Taken together, these data demonstrate clearly that inhibition of
inflammatory chemokines and their cognate chemokine receptors is a
major consequence of TCR vaccination that undoubtedly contributes to
reduced inflammation in the primary target organ, the SC, and
protection from paralytic signs of EAE.
Continued expression of chemokines with reduced numbers of infiltrating mononuclear cells in the brain of TCR-protected mice further supports the notion that chemokine production is not simply correlated with the degree of cellular infiltration. In a previous study using cotransferred encephalitogenic and TCR-reactive T cells (31), we demonstrated that the regulatory T cells prevented activation and infiltration of pathogenic T cells into the CNS. Moreover, the regulatory T cells themselves migrated into the CNS and appeared to selectively inhibit further recruitment of nonpathogenic CD4+ T cells, with little effect on other cell types. Taken together, these observations suggest that TCR vaccination may have dual effects: 1) systemic inhibition of pathogenic T cell activation that limits migration across the endothelial barrier; and 2) direct effects within the CNS of infiltrating TCR-reactive T cells, including local inhibition of pathogenic and bystander Th1 cells (31), and possibly direct modulation of chemokine expression by other infiltrating cells and resident tissue cells. The latter effect may occur more readily in brain, where increased expression of Th2 associated chemokine receptors, CCR3 and CCR4 (30, 32, 33), was detected.
Recent studies have demonstrated that RANTES and MCP-1 play a role in
both activating and recruiting leukocytes, particularly activated
memory T cells (15, 34, 35). IP-10, a member of the CXC
family, has also been reported to be chemotactic for macrophages and T
cells (36, 37). Several groups have demonstrated elevated
expression of RANTES, IP-10, and MCP-1 in the SC of mice at the peak of
EAE (13, 14, 15, 17). Moreover, RANTES, IP-10, and MIP-1,
were found in MS lesions, and cognate receptors, CCR5 and CXCR3, were
found on MS T cells (21, 22, 38). Our study confirms the
predominance of RANTES, IP-10, and MCP-1 in the SC of mice with acute
EAE, as well as the less pronounced expression of MIP-ß and MIP-1.
However, in contrast with Godiska et al. (15), we also
detected message for MIP-2 but not TCA3 in SC, due possibly to
differences in the murine EAE models used. Previous studies by the
Karpus group using Abs to inhibit chemokine function have demonstrated
the importance of MIP-1 in acute EAE and MCP-1 in relapsing EAE, but
neutralization of RANTES had no effect on disease expression (18, 19). Other studies in EAE and experimental autoimmune neuritis
(EAN) have shown that MIP-2 production did not correlate with disease
severity (14, 39). TCR vaccination profoundly reduced
expression of both MIP-1 and MCP-1 in SC, thus providing a plausible
explanation for the almost complete protection against EAE.
In summary, our study demonstrated highly up-regulated mRNA expression
of inflammatory chemokines in the affected target organ during the
acute phase of paralytic EAE that was strongly inhibited in
TCR-protected mice. TCR vaccination induces regulatory T cells that
overexpress IL-4 and IL-10 and can inhibit the activation, production
of IFN-, and ability to transfer EAE by encephalitogenic Th1 cells
specific for MBP-NAc1–11 peptide (11, 31). TCR-induced protection may thus be mediated in part by
local production of Th2 cytokines by TCR-specific T cells in CNS,
possibly inhibiting production of chemokines and their associated
receptors. The interactions between cytokines and chemokines and
chemokine receptors is an area of intense interest and will be the
subject of subsequent studies using purified encephalitogenic and
regulatory T cell populations.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Agata Matejuk, R&D-31, VA Medical Center, 3710 S.W. U.S. Veterans Hospital Road, Portland, OR 97201. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MS, multiple sclerosis; Ltn, lymphotactin; MIP-1ß, macrophage-inflammatory protein-1ß; MIP-1, macrophage-inflammatory protein-1; MIP-2, macrophage-inflammatory protein-2; IP-10, IFN-inducible 10-kDa protein; MCP-1, monocyte chemoattractant protein-1; TCA-3, T cell activation Ag; LN, lymph node; Tg, transgenic; SC, spinal cord; RPA, RNase protection assay.
Received for publication October 4, 1999. Accepted for publication January 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and monocyte chemoattractant peptide-1 elicit immediate and late cutaneous reactions and activate murine mast cells in vivo. J. Immunol. 152:1298.[Abstract]
in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
and MIP-1ß in rat experimental autoimmune encephalomyelitis by in situ hybridization. J. Neuroimmunol. 77:17.[Medline]
- and ß-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am. J. Pathol. 150:617.[Abstract]
and MCP-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as Th1/Th2 lymphocyte differentiation. J. Leukocyte Biol. 62:681.[Abstract]
and monocyte chemotactic protein-1. J. Neuroimmunol. 92:98.[Medline]
and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. USA 96:6873., MCP-1 and MCP-2 and potential role of neutralization of these chemokines in the regulation of immune responses during experimental autoimmune neuritis in Lewis rats. J. Neuroimmunol. 98:168.[Medline]
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