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* Department of Clinical Neuroscience, Neuroimmunology Unit, Karolinska Institutet, Stockholm, Sweden;
Department of Neurology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA 02115;
Department of Neurology, Karolinska Hospital, Stockholm, Sweden; and
Department of Neurology, Danderyd Hospital, Stockholm, Sweden
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Abstract |
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mRNA in cerebrospinal fluid mononuclear cells. In contrast, expression of TIM-3 correlated well with high expression of IFN- and TNF-
. These data imply the differential expression of human TIM molecules by Th1 and Th2 cells and may suggest their differential involvement in different phases of a human autoimmune disease. |
Introduction |
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The TIM family may be of relevance for organ-specific inflammatory diseases for a number of reasons. Tapr, a genetic locus regulating susceptibility for asthma/airway hyperreactivity and T cell expression of IL-4 and IL-13, was recently mapped to the Tim locus, and it was suggested that polymorphisms in the TIM-1 gene may associate with the phenotype (4). Conversely, TIM-3 has been shown to be expressed on effector Th1 cells in mice (4, 5). In addition, administration of an anti-TIM-3 Ab in vivo exacerbated the clinical course of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice with associated activation and expansion of macrophages (5).
The TIM family of genes may thus be considered candidates for regulating human autoimmune and allergic diseases. Although polymorphisms in the TIM-1 and TIM-3 genes have been described in mice in association with disease susceptibility (4), a linkage with polymorphic alleles of TIM-1 with human asthma has recently been reported (8, 9). The potential linkage of genetic polymorphisms in the human TIM genes with susceptibility to human Th1-related diseases such as multiple sclerosis (MS) largly remains to be explored. Although they are differentially expressed on murine Th1 and Th2 cells, whether TIM molecules are differentially expressed on human Th1 and Th2 cells has not been reported. Furthermore, whether the expression of TIM molecules correlates with disease activity and cytokine levels in vivo has also not been studied.
In this study, we examined the expression of TIM-1 and TIM-3 on specific Th1 and Th2 human cell lines in vitro and in a human autoimmune disease, MS. Th1 lines expressed higher TIM-3 mRNA, whereas Th2 lines were characterized by higher TIM-1 expression. Moreover, TIM-1 mRNA was significantly up-regulated in cerebrospinal fluid mononuclear cells (CSF-MC) of patients with MS. The increased expression of TIM-1 was associated with inactive clinical disease. In addition, while expression of TIM-3 in the CSF correlated well with the mRNA expression of IFN- and TNF-, high levels of TIM-1 were associated with low expression of IFN- in CSF-MC. These data suggest that TIM molecules are differentially expressed on human Th1 and Th2 T cells, and are functionally involved during the course of a human autoimmune disease.
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Materials and Methods |
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PBMC of a healthy subject were repeatedly restimulated with a random copolymer, glatiramer acetate (Copaxone; Teva Marion Partners, Kansas City, MO), to establish TCL. Briefly, PBMC were isolated by Ficoll-Hypaque gradient centrifugation. Cells were maintained in complete RPMI 1640 medium with 5% human AB serum (Omega Scientific, Tarzana, CA), and 1.5 x 103 cells/well were stimulated with either 40 or 100 µg/ml glatiramer acetate in 96-well microtiter plates. To generate Th1 and Th2 lines, 10 ng/ml human IL-12 (R&D Systems, Minneapolis, MN) and 1 µg/ml anti-IL-4 (R&D Systems) or 10 ng/ml IL-4 (R&D Systems) and 10 µg/ml anti-IL-12 (R&D Systems) Abs were added, respectively. Human rIL-2 was added at 10 U/ml concentration 5 days later. Reactivity to Ag and cytokine production was tested on day 11–12 by standard proliferation assay and ELISA. Cells were restimulated with glatiramer acetate every 14 days.
Patients and controls
The MS population consisted of 77 subjects (mean age 41.6 years, range 17–65 years; 53 females and 24 males; 72 with and 5 without IgG oligoclonal bands in CSF), fulfilling the McDonald criteria of MS (10). Blood samples were available from all patients with MS and paired CSF samples from 61 of them. Fifty-seven patients displayed relapsing remitting (RRMS), 17 secondary progressive (SPMS), and 3 primary progressive disease courses. In the RRMS group, 9 CSF samples were collected during relapse and 35 during remission.
In addition, 10 patients with clinically isolated syndrome (CIS), who had a first clinical relapse with one or more magnetic resonance imaging (MRI) lesions characteristic to MS (10), were also sampled as a separate group. The noninflammatory control (NIC) group consisted of 75 subjects (mean age 39.2 years, range 14–74 years; 55 females and 20 males; all without IgG oligoclonal bands in CSF) with the following diagnoses: unspecified sensory disturbance (n = 38), idiopathic intracranial hypertension (n = 6), unspecified headache (n = 6), migraine (n = 3), cervicalgia (n = 2), chronic idiopathic pain (n = 2), amyotrophic lateral sclerosis (n = 2), vestibular disorder (n = 1), rhizopathy (n = 1), chronic idiopathic fatigue (n = 1), noninflammatory myelopathy (n = 1), noninflammatory peripheral neuropathy (n = 1), as well as healthy individuals (n = 11).
Blood samples were available from all NIC and paired CSF samples from 67 subjects. No subject had received immunomodulatory treatment (corticosteroids, IFN-
, glatirameracetate, Igs, or cytostatic drugs) during the 3-mo period before sampling. The local ethical committee approved the study.
Preparation of PBMC and CSF-MC
Peripheral blood was sampled in sodium citrate-containing cell preparation tubes (Vacutainer CPT; BD Biosciences, San Jose, CA) and CSF in siliconized glass tubes. CSF samples were immediately centrifuged, and the pellet was recovered and stored at –70°C until use. PBMC were separated by density gradient centrifugation. Cells from the interphase were collected and washed twice with Dulbecco's PBS. The proportion of viable cells was assessed by trypan blue exclusion. More than 95% of the cells were viable. Finally, the cells were pelleted, frozen on dry ice, and stored at –70°C until use.
Cytokine measurement by ELISA
Supernatants of TCL were collected after 48 h of restimulation with glatiramer acetate and stored at –70°C. Secretion of IFN- and IL-13 was measured by ELISA. Briefly, cytokine Abs for IL-13 (BD Biosciences) and IFN- (Endogen, Rockford, IL) were coated to 96-well Immulon 4HBX plates (Thermo Labsystems, Franklin, MA) at a concentration of 1 µg/ml at 4°C overnight. The plates were washed and treated with PBS containing 1% BSA (Sigma-Aldrich, St. Louis, MO), followed by overnight incubation at 4°C with culture supernatants and with cytokine standards (human rIFN-, Life Technologies, Rockville, MD; human rIL-13, R&D Systems, Minneapolis, MN). The plates were washed and incubated with their corresponding biotinylated anti-cytokine-detecting mAb (1 µg/ml) for 2 h (anti-human IFN-, Endogen; anti-human IL-13, BD Biosciences). They were developed after adding avidin peroxidase (Sigma-Aldrich) and SureBlue, TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Absorbance was measured using ELISA reader (Bio-Rad, Melville, NY) at 450 nm, a standard curve for each assay being generated and cytokine production calculated. Assays were performed in duplicate.
Relative quantitation of mRNAs by real-time quantitative RT-PCR
Cell pellets were lysed, and total RNA was extracted (Qiagen total RNA extraction kit, Hilden, Germany). Samples were incubated with 27 kU of DNase (Qiagen; RNase-free DNase set) for 30 min at 37°C to avoid amplification/detection of contaminating genomic DNA. Reverse transcription was performed with 10 µl of total RNA, random hexamer primers (0.1 µg; Life Technologies), and Superscript reverse transcriptase (200 U; Life Technologies). Amplification was performed using an ABI PRISM 7700 Sequence Detection System (PerkinElmer, Norwalk, CT) using the 5'-nuclease method (TaqMan) with a two-step PCR protocol (95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min).
All primers and probes were designed with the Primer Express Software (PerkinElmer) in our laboratory, except for 18S rRNA (PerkinElmer). The TaqMan probes were labeled with FAM as reporter dye and TAMRA as quencher dye, except for the 18S rRNA probe, which was labeled with VIC as reporter dye and TAMRA as quencher dye. The sequences of all primers and probes used in this study are as follows. The TIM-1 sequences are: FAM probe, 5'-CTG TCT TGG TGC TTC TTG CTC TTT TGG GT-3'; forward primer, 5'-GAA CAT AGT CTA CTG ACG GCC AAT AC-3'; reverse primer, 5'-GAA CCT CCT TTT TGA AGA AAT ACT TTT T-3'. The TIM-3 sequences are: FAM probe, 5'-ACA TGG CCC AGC AGA GAC ACA GAC ACT-3'; forward primer, 5'-TCC AAG GAT GCT TAC CAC CAG-3'; reverse primer, 5'-GCC AAT GTG GAT ATT TGT GTT AGA TT-3'. The TNF- sequences are: FAM probe, 5'-CTC TGG CCC AGG CAG TCA GAT CAT CTT-3'; forward primer, 5'-CCA GGG ACC TCT CTC TAA TCA GC-3'; reverse primer, 5'-CTC AGC TTG AGG GTT TGC TAC A-3'. The IFN- sequences are: FAM probe, 5'-TTG AAG AAT TGG AAA GAG GAG AGT GAC AGA AAA ATA-3'; forward primer, 5'-ATA TTT TAA TGC AGG TCA TTC AGA TGT AG-3'; reverse primer, 5'-TGA AGT AAA AGG AGA CAA TTT GGC T-3'. The IL-10 sequences are: FAM probe, 5'-CCC TGT GAA AAC AAG AGC AAG GCC G-3'; forward primer, 5'-CGG CGC TGT CAT CGA TTT-3'; reverse primer, 5'-TTA AAG GCA TTC TTC ACC TGC TC-3'. The IL-4 sequences are: FAM probe, 5'-TGC ACC GAG TTG ACC GTA ACA GAC ATC TT-3'; forward primer, 5'-GCA CAA GCA GCT GAT CCG AT-3'; reverse primer, 5'-CAG GAA TTC AAG CCC GCC-3'. The GAPDH sequences are: FAM probe, 5'-TAT TGT TGC CAT CAA TGA CCC CTT CAT TGA C-3'; forward primer, 5'-AGG GCT GCT TTT AAC TCT GGT AAA-3'; reverse primer, 5'-CAT ATT GGA ACA TGT AAA CCA TGT AGT TG-3'. The 18S rRNA sequences are: VIC probe, 5'-TGC TGG CAC CAG ACT TGC CCT C-3'; forward primer, 5'-CGG CTA CCA CAT CCA AGG AA-3'; reverse primer, 5'-GCT GGA ATT ACC GCG GCT-3'.
Amplification/Detection of contaminating genomic DNA was avoided by constructing one of the primers over an exon/intron boundary. In preliminary experiments, the primer pairs had been tested using a conventional PCR protocol. The PCR products were run in an agarose gel and were in all cases confined to a single band of the expected size. Sequencing of the different bands (Cybergene AB, Huddinge, Sweden) confirmed homology with the reported sequences for human GAPDH, TIM-1, TIM-3, TNF-, IFN-, IL-10, and IL-4, respectively. Relative quantitation of mRNA levels was performed using the standard curve method, with amplification of mRNA and 18S rRNA or GAPDH in separate tubes (described in detail in User Bulletin 2; PerkinElmer; Applied Biosystems, Branchburg, NJ, 1997). The standard curves were created using five serial dilutions (1:10, 1:102, 1:103, 1:104, and 1:105) of cDNA from human blood cells stimulated with either staphylococcal enterotoxin B or PHA. The samples were run in duplicate with primers and probes against 18S rRNA or GAPDH and the target mRNA in different wells. Samples without added cDNA served as negative controls. The relative amount of mRNA in each sample was calculated as the ratio between the target mRNA and the corresponding endogenous control, 18S rRNA, or GAPDH. The ratio for each target was normalized to an arbitrary maximum value of 1.0 represented by the sample with highest ratio.
Relative quantitation of TIM-1 and TIM-3 mRNA expressed by TCL
Glatiramer acetate-specific Th0/Th1 and Th2 cell lines were harvested after 6-h stimulation with soluble 2.5 µg/ml anti-CD28 and plate-bound 0.5 µg/ml anti-CD3. Total RNA was isolated from 5 x 106 cells using RNeasy Mini Kit (Qiagen). To eliminate genomic DNA contamination, samples were digested with DNase I (Life Technologies). A quantity amounting to 2–3 µg of total RNA was reverse transcribed into cDNA using the Taqman Reverse Transcriptase Reagents Kit (Applied Biosystems). To quantify TIM-1 and TIM-3 RNA, multiplex TaqMan RT-PCR was applied using VIC-TAMRA-labeled GAPDH control primers and probe mixture (Applied Biosystems) as internal control. PCR were run in duplicate, and relative expression of template compared with GAPDH was calculated as 
Ct (difference in cycle threshold value of TIM and GAPDH) (Table I). The relative expression for each target was then normalized to an arbitrary maximum value of 1.0 represented by the sample with lowest Ct (highest expression).
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Blood samples were collected from patients with MS (n = 8) and NIC (n = 4). PBMC were isolated, as described above. mAbs were purchased from BD Biosciences and titrated to optimal concentrations. Cell suspensions were labeled with the following anti-human mAbs in different combinations: FITC-conjugated anti-CD3 and anti-CD14, PE-conjugated anti-CD8 and anti-CD20, and allophycocyanin-conjugated CD4. The Simultest CD3/CD16 + CD56 was used for the detection of NK cells. The samples were incubated in dark at 4°C for 20 min, washed twice with PBS supplemented with 0.09% NaN3 and 3% FCS, and resuspended in 1 ml of PBS before analysis using a MoFlo high-speed cell sorter (Dakocytomation, Glostrup, Denmark). One region (R1) was defined in the light scatter plot corresponding to live cells, and another region (R2) delineated cells positive for the respective Ab. Cell sorting was performed with the sorting gate set as R1 x R2. Approximately 2–10 x 105 cells of each subpopulation were sorted into 2-ml Eppendorf tubes. After centrifugation, the cell pellets were kept at –70°C until RNA extraction. Sorting purity was determined to be >95%.
Statistical analysis
Differences in relative mRNA levels of TIM-1, TIM-3, and cytokines in CSF-MC, PBMC, and TCL were tested for significance with the nonparametric Wilcoxon signed ranks test (JMP 3.2; SAS Institute, Cary, NC). Correlations between TIM and cytokine mRNA levels were analyzed with Spearman's rank test (GraphPad Prism 3.0, San Diego, CA). The RRMS samples were divided into two arbitrarily defined groups: low relative expression 0–0.05 and high relative expression >0.05. Differences between samples taken during relapses and remission were then evaluated with Fisher's exact test (JMP 3.2).
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Results |
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Recent data have suggested the association of the TIM-3 molecule with Th1-related T cell responses and disease in mice (5). In addition, TIM-1 alleles have been implicated in susceptibility to Th2-related airway hyperreactivity in mice (4), although TIM-1 expression on Th2 cells has not been formally demonstrated. Furthermore, there are no available data on the expression of TIM molecules on differentiated Th1 and Th2 cells in humans.
To examine the expression of TIM-1 and TIM-3 by human Th1 and Th2 cells, we used a random copolymer, glatiramer acetate, to establish a panel of MHC class II-restricted CD4 cells from both naive and memory T cells differentiated under Th1- and Th2-polarizing conditions. As depicted in Fig. 1, A and B, and Tables I and II, we established seven Th1 cell lines that secreted significant amounts of IFN-, and generated 25 Th2 lines that produced significant levels of IL-13. Expression of TIM mRNAs was analyzed by real-time RT-PCR after four to six rounds of polarization following a 6-h in vitro stimulation with anti-CD3 and anti-CD28 mAbs. Although TIM-3 mRNA was highly expressed on stimulated Th1 cells, the expression of TIM-1 was much lower, indicated by higher Ct values representing difference in cycle threshold values of GAPDH and TIM-1 (Tables I and II). More importantly, TCL expressing low or no IL-13 and high IFN- (Th1/Th0) were characterized by significantly higher expression of TIM-3 (p < 0.001) (Fig. 1D), and T cells secreting low IFN- and high IL-13 (Th2) were characterized by significantly higher expression of TIM-1 (p < 0.001) (Fig. 1C). Although the sample size was too small to correlate TIM-3 expression with amount of IFN-/IL-13 production of individual TCL, it is noteworthy that the two Th0 lines that produced large amounts of IL-13 with IFN- expressed the lowest TIM-3 mRNA (1.4 and 1.5 in Table I). In contrast, expression of TIM-1 mRNA appeared to associate with higher IL-13 and a trend toward low IFN- secretion (Tables I and II).
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Because TIM-1 and TIM-3 appeared to be differentially expressed in human Th2 and Th1 lines, respectively, we next analyzed the relative expression of these molecules in PBMC and CSF-MC of patients with MS, a presumed autoimmune disease of the CNS. Similar to the EAE model, several lines of evidence suggest that MS may also be mediated by autoreactive Th1 cells (11).
Using real-time RT-PCR, we first examined the expression of TIM-1 and TIM-3 mRNA in PBMC of 61 patients with MS and 67 controls (NIC). Although TIM-1 and TIM-3 mRNA expression was detected in all PBMC samples, there was no significant difference in the relative expression of either molecules when comparing patients with MS to NIC (Fig. 2, A and B).
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Although TIM-3 mRNA expression was detected in all CSF-MC samples from the different groups, its relative expression was not significantly higher in patients with MS compared with NIC. It should be noted, however, that expression of TIM-3 was more heterogeneous in MS and a portion of patients expressed high level of TIM-3 (Fig. 2D). Collectively, these data suggest that expression of TIM-1 is increased in the CNS of patients with MS.
TIM expression in patients with CIS, RRMS, and SPMS
Because TIM-1 and TIM-3 are expressed on Th2 and Th1 clones/lines, respectively, we reasoned that these molecules might be differently expressed during acute clinical events as compared with phases of the disease during which no clinical event was evident. We therefore isolated CSF-MC of 9 patients during clinical relapses and 35 patients during remissions. Although all patients with clinical relapses had very low expression levels of TIM-1 in the CSF, samples collected during clinical remission ranged from low to high expression. A significantly higher number of patients had high TIM-1 mRNA in the CSF during inactive disease than during acute clinical events (p = 0.016). This indicated that the observed up-regulation of TIM-1 mRNA in CSF-MC of MS might be related to clinically inactive phases of the disease (Fig. 3A). Expression of TIM-3 was not significantly different comparing clinical remission with relapse samples (data not included).
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We also contemplated that expression of TIM molecules might be dynamically different in early and late phases of the disease. Although CIS is considered a pre-MS disease with a high conversion rate into definitive MS, the majority of patients with RRMS convert to a secondary chronic progressive phase of the clinical spectrum. We thus analyzed CSF of 9 patients with clinically inactive CIS and 17 patients with SPMS. The percentage of patients with CIS with TIM-1 mRNA expression in CSF-MC was very high (89%), similar to MS (85%). Similar data were obtained from the SPMS patients who did not experience clinical relapses, in whom levels of TIM-1 in CSF-MC were comparable to RRMS in remission (Fig. 3B).
In summary, these data suggest that the elevated level of TIM-1 in the CSF is associated with the inactive phases of the disease when no clinical events were evident. Moreover, expression of TIM-1 was similarly elevated in both CIS and SPMS patients, suggesting that it might have a steady expression in both the early, pre-MS and the late secondary progressive phases of the disease.
The cellular source of TIM-1 and TIM-3 expression
TIM molecules are expressed by T cells in mice, but there is a possibility that other cell types also express TIM-1 and TIM-3 mRNA in humans. To analyze the cellular source of TIM, PBMC from patients with MS and NIC were FACS sorted into the following populations: CD3+CD4+ (CD4+ T cells), CD3+CD8+ (CD8+ T cells), CD14+ (monocytes), CD20+ (B cells), CD3–CD16+CD56+ (NK cells), and T cells with NK markers (CD3+CD16+CD56+). TIM-1 mRNA was expressed by CD4+ T–, CD8+ T–, NK–, and CD3+CD16+CD56+ (NKT) cells, whereas TIM-1 was undetectable in monocytes and B cells (Fig. 4A). The major sources of TIM-3 mRNA in the PBMC were NK- and NKT cells (Fig. 4B), while much lower levels were present in all other sorted populations, including CD4+ T cells. The expression patterns of TIM-1 and TIM-3 were similar in both MS patients and NIC, and the distribution of the cell populations reflected the expected distribution of these cell types in human PBMC (data not included).
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Although we observed a differential expression of TIM-1 and TIM-3 by Th1 and Th2 lines in vitro, the in vivo correlation of cytokines and TIM molecules has never been examined during a disease process in either mice or humans. We therefore attempted to examine whether our PBMC and CSF-MC samples from MS and NIC differ in the expression of Th1 and Th2 cytokines, and whether there was a correlation between the expression of cytokines and TIM molecules in MS.
First, we analyzed the expression of Th1 and Th2 cytokines in the PBMC and CSF-MC of MS patients and NIC. There was a significant increase in the relative expression of IFN- (Fig. 5A) and IL-10 (Fig. 5C) mRNAs in both PBMC and CSF-MC obtained from patients with MS compared with NIC. Although expression levels of TNF- mRNA (Fig. 5B) were not different in PBMC of MS and controls, it was significantly higher in CSF-MC of patients with MS. IL-4 mRNA was detected in PBMC of both MS patients and controls, but no significant difference between the groups was discernible. Levels of IL-4 mRNA in CSF-MC were below the detection limit (data not included).
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, TNF-, or IL-10 in CSF-MC (data not included). However, when patients without acute clinical events were separately analyzed, high expression of TIM-1 in the CSF-MC was associated with low IFN- message (Fig. 6A). No correlation with another inflammatory cytokine, TNF- mRNA expression was evident (Fig. 6B). In addition, high expression of TIM-1 mRNA on CSF-MC correlated well with high expression of IL-10 mRNA in clinically inactive phases of the disease (Fig. 6C).
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(Fig. 7A), TNF- (Fig. 7B), and IL-10 (Fig. 7C) in CSF-MC. Although the sample size of patients in acute clinical relapse was low, we observed a trend for association of high TIM-3 expression with high expression of IFN- and TNF- in CSF-MC during acute clinical events (data not included).
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Discussion |
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. In contrast, expression of TIM-3 in the CSF correlated well with the expression of the inflammatory cytokines IFN- and TNF-. We found that human Th1/Th0 lines expressed higher TIM-3 mRNA, while Th2 lines were characterized by TIM-1 expression, suggesting the differential expression of these molecules on human Th1 and Th2 lines. Th0 lines expressed lower TIM-3 mRNA compared with polarized Th1 lines, underscoring a role of this molecule in Th1 differentiation, as previously reported for murine T cells (5). Compared with TIM-3, we generally observed a lower expression of TIM-1 by all activated T cells. Because our positive controls consisting of human TIM-1 transfectants expressed mRNA very efficiently, we do not think that this was related to a less efficient PCR amplification. Either optimal conditions for up-regulation of TIM-1 mRNA may differ from requirements for TIM-3 expression or TIM-1 is expressed at very low level in human cells.
In human MS, we found that the number of individuals expressing high levels of TIM-1 in CSF-MC was significantly increased among patients with MS compared with control individuals, although there was no difference in the expression of TIM molecules in PBMC. Because MS is considered to be an autoimmune disease of the CNS, it is possible that cells expressing high TIM-1 are specifically enriched in the CNS compartment. Moreover, when patients were grouped according to the clinical activity of the disease, the difference observed in the expression of TIM-1 was mainly related to patients in clinically inactive phases of MS. It thus seems that TIM-1-expressing T cells, which might be of Th2 phenotype, are increased in the clinical remission phase of disease and might be involved in an anti-inflammatory response. Alternatively, expression of TIM-1 on CSF-MC might be down-regulated during acute clinical events. Alterations in the NK or NK T cell population could also explain this finding, as discussed below. Because we observed a bimodal expression of TIM-1 among patients without acute clinical events, we also considered the possibility that patients with low expression of TIM-1 might have a more active disease. Indeed, patients without acute clinical events, but expressing low TIM-1 on CSF-MC, had a tendency to have more active disease, as indicated by higher numbers of MRI lesions and higher CSF-IgG indexes, or more advanced disease as characterized by higher EDSS.
We also found that expression of TIM-1 was higher in patients with CIS (pre-MS) who had experienced only a single clinical relapse, but also in SPMS, a conversion of RRMS into a nonundulant, progressive course in which axonal pathology might be responsible for progression of the disease (16). Because these two groups of patients represent two ends of the clinical spectrum, it may suggest a relatively steady expression of TIM-1 during the evolution of disease.
In contrast to expression of TIM-1, we could not record any difference in the expression of TIM-3 between patients with MS and NIC, neither in PBMC nor in CSF-MC. However, TIM-3 is mainly expressed by human Th1 clones in vitro, which are known to be prone to apoptosis, and may die quickly in the CNS (17, 18, 19). In addition, the expression of TIM-3 mRNA in mice peaked just before onset of EAE in lymph nodes, and right at the onset of disease in the brain, and was then down-regulated or diluted to near basal levels as disease progressed (5). Thus, expression profiles of TIM-3 and TIM-1 mRNA in the CSF might be very different in MS, and we may expect only a short period of increased TIM-3 expression in contrast to the more steady expression of TIM-1. We should also consider that the activation requirements for the expression of TIM-1 and TIM-3 might be very different, as indicated by the in vitro data.
An additional level of complexity was added by the fact that TIM-1 and TIM-3 mRNAs were expressed in several different leukocyte populations in the PBMC. TIM-1 mRNA was expressed in CD4+, CD8+, NK, and NKT cells, whereas TIM-3 mRNA was expressed in many different cell types, but predominantly in NK cells ex vivo. There are no reports of NK cell expression of TIM-1 or TIM-3 in murine models to date. Further single cell studies may determine whether expression of TIM-3 is associated with only a small subset of NK and NKT cells producing Th1 cytokines, or whether TIM-3 represents an intrinsic marker for these populations. Although the expression of TIM-3 was lower on CD4+ and CD8+ T cells ex vivo, we have to consider that TIM-3 is expressed on highly differentiated and terminally committed Th1 cells, and that their activation might be strictly regulated, resulting in the inhibition of Th1 cells expressing TIM-3 on the surface. This is consistent with recent data reporting that blockade of TIM-3/TIM-3 ligand results in dramatic expansion of Th1 cells, thus suggesting that TIM-3 pathway is involved in inhibition of Th1 responses (20). Alternatively, TIM-3-expressing T cells might be differently compartmentalized or may only up-regulate TIM-3 for a short period of time.
Besides the unique advantage of analyzing these cell surface molecules ex vivo and examining the dynamics of activation related to the disease activity, MS materials also provided the opportunity to correlate the expression of TIM-1 and TIM-3 with Th1 and Th2 cytokine levels ex vivo. We found that expression of TIM-3 strongly correlated with the expression of the prototypic Th1 cytokine IFN- in CSF-MC, supporting our in vitro data. We also found a correlation with the expression of another inflammatory cytokine, TNF-. In contrast, the high TIM-1 mRNA expression observed in inactive clinical MS was associated with low expression of IFN-, suggesting that CSF-MC express an elevated level of TIM-1, but low levels of IFN- during the clinically inactive phase of the disease. Both TIM-1 and TIM-3 expression correlated with the expression of IL-10, but the association of this cytokine with Th1 and Th2 conditions is not clear in humans (21, 22). The possibility of detecting any Th2-type cytokine associations with TIM-1 was hampered by the failure to detect IL-4 in CSF.
The findings of elevated TIM-1 mRNA levels in MS, a correlation between TIM-3 and IFN- expression, and the differential expression by human Th1 and Th2 cells all support the notion that these molecules are indeed expressed differentially on human Th1 and Th2 cells and are also involved in regulating their effector functions. However, we have to consider that our ex vivo data did not reflect the expression of these molecules at the single-cell level, and thus we cannot conclude whether expression of TIMs and cytokines by single cells is responsible for the observed correlations. It is also not clear presently how TIM molecules may contribute to the disease process.
In summary, we demonstrate the differential in vitro expression of TIM-1 and TIM-3 by human Th2 and Th1/Th0 cells, respectively. Accordingly, we also show that expression of TIM-3 correlated well ex vivo with the expression of inflammatory Th1 cytokines in the CNS compartment of patients with MS. Conversely, the Th2-related molecule TIM-1 was increased on the CSF-MC of patients in the inactive, but not in the clinically active phase of the disease and was associated with low IFN- expression, suggesting its connection with disease regulation. Interestingly, the cellular sources of the TIMs were diverse and included NK cells, NK T cells, CD8+ T cells, and CD4+ T cells. Together, these data provide the first evidence for the differential expression of human TIM-1 and TIM-3 in relation to Th1 and Th2 cytokines in vitro and in an autoimmune disease, MS, ex vivo. Our data imply that further studies to explore the polymorphisms of TIM-1 and TIM-3 in human autoimmune and allergic diseases may provide valuable surrogate markers for disease activity and potential susceptibility alleles for disease, as has recently been shown for human atopy (9).
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Acknowledgments |
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Footnotes |
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2 M.K. and Z.I. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Mohsen Khademi, Neuroimmunology Unit, CMM, L8:04, Karolinska Hospital, SE 171 76 Stockholm Sweden. E-mail address: mohsen.khademi{at}cmm.ki.se
4 Abbreviations used in this paper: TIM, T cell Ig- and mucin-domain-containing molecule; CIS, clinically isolated syndrome; CSF, cerebrospinal fluid; CSF-MC, CSF mononuclear cell; Ct, difference in cycle threshold value; EAE, experimental autoimmune encephalomyelitis; EDSS, expanded disability status scale; MRI, magnetic resonance imaging; MS, multiple sclerosis; NIC, noninflammatory control; RRMS, relapsing remitting MS; SPMS, secondary progressive MS; TCL, T cell line.
Received for publication January 16, 2004. Accepted for publication March 22, 2004.
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References |
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- and interleukin-4-secreting cells in multiple sclerosis. J. Neuroimmunol. 46:123.[Medline]
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L. L. Binne, M. L. Scott, and P. D. Rennert Human TIM-1 Associates with the TCR Complex and Up-Regulates T Cell Activation Signals J. Immunol., April 1, 2007; 178(7): 4342 - 4350. [Abstract] [Full Text] [PDF]
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S. Frisancho-Kiss, J. F. Nyland, S. E. Davis, M. A. Barrett, S. J. L. Gatewood, D. B. Njoku, D. Cihakova, E. K. Silbergeld, N. R. Rose, and D. Fairweather Cutting Edge: T Cell Ig Mucin-3 Reduces Inflammatory Heart Disease by Increasing CTLA-4 during Innate Immunity. J. Immunol., June 1, 2006; 176(11): 6411 - 6415. [Abstract] [Full Text] [PDF]
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H. Geng, G.-M. Zhang, D. Li, H. Zhang, Y. Yuan, H.-G. Zhu, H. Xiao, L.-F. Han, and Z.-H. Feng Soluble Form of T Cell Ig Mucin 3 Is an Inhibitory Molecule in T Cell-Mediated Immune Response J. Immunol., February 1, 2006; 176(3): 1411 - 1420. [Abstract] [Full Text] [PDF]
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T. T. Chen, L. Li, D.-H. Chung, C. D.C. Allen, S. V. Torti, F. M. Torti, J. G. Cyster, C.-Y. Chen, F. M. Brodsky, E. C. Niemi, et al. TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis J. Exp. Med., October 3, 2005; 202(7): 955 - 965. [Abstract] [Full Text] [PDF]
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