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Frequency of blood CX₃CR1-positive natural killer cells correlates with disease activity in multiple sclerosis patients

Carmen Infante-Duarte^*, Alexandra Weber^*, Jörn Krätzschmar, Timour Prozorovski^*, Susan Pikol^*, Isabell Hamann^*, Judith Bellmann-Strobl^*, Orhan Aktas^*, Jan Dörr^*, Jens Wuerfel^*, Claus-Steffen Stürzebecher and Frauke Zipp^*,1

^* Institute of Neuroimmunology, Charité University Hospital Berlin, Germany;
Departments of Genomics and Bioinformatics, and
Department of Pharmacogenomics, Schering AG, Berlin, Germany

¹ Correspondence: Institute of Neuroimmunology, Neuroscience Research Center, Charité University Hospital, Berlin 10098, Germany. E-mail: frauke.zipp{at}charite.de

SPECIFIC AIMS

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by enormous variability in its clinical presentation and course, and in which clear diagnostic parameters are lacking. The aims of our study were: 1) to identify new cellular pathways involved in the pathogenesis of multiple sclerosis and 2) to define blood markers that would facilitate the diagnosis of the disease and of its clinical forms by performing a genomic analysis of MS patients and healthy individuals.

PRINCIPAL FINDINGS

1. Differentially regulated genes in MS patients
Employing oligonucleotide arrays, we identified 17 genes that were differentially expressed in MS patients compared with healthy individuals. Genes with increased expression were involved in: 1) cell cycle and activation; 2) chemotaxis, adhesion and transendothelial migration; and 3) intracellular transport mechanisms and other cellular processes. Genes with decreased expression were involved in the response of monocytes to interferons and in the regulation of B cell response. Of 16 genes differentially regulated in relapsing-remitting (RRMS) patients, six were similarly expressed in primary-progressive (PPMS). We did not identify any genes that would provide a distinction between RRMS and PPMS patients.

2. Decreased CX₃CR1 gene and protein expression in MS patients
The data obtained using microarray analysis indicated that expression of the chemokine receptor CX₃CR1 is decreased in both RRMS and PPMS when compared with healthy donors (HD). The mean expression of the receptor in RRMS and PPMS relative to its expression in HD was –4.9 and –3.3 respectively (Fig. 1 A). To confirm these results, we examined gene expression of CX₃CR1 in expanded patient and HD cohorts using real-time RT-PCR and protein expression using flow cytometric analysis. The data plotted in Fig. 1B, C (mean relative expression±SEM) confirm that MS patients showed a reduced CX₃CR1 gene (Fig. 1B ) and protein (Fig. 1C ) expression compared with control individuals. Gene expression was reduced by 40% and 57% in peripheral blood mononuclear cells (PBMC) from RRMS and PPMS patients respectively when compared with HD (Fig. 1B ).

View larger version (22K): [in this window] [in a new window]   Figure 1. CX3CR1 expression in peripheral blood mononuclear cells from patients with MS. A) The diagrams display the results of CX3CR1 gene expression obtained from the microarray analysis of 10 RRMS (left) and 8 PPMS patients (right), respectively. For each patient, the mean expression of the 12 pairwise comparisons (the particular patient vs. the 12 healthy controls) is shown. The hatched bars to the right of each diagram represent the mean expression of the 10 RRMS and 8 PPMS patients respectively. B) Gene expression of CX3CR1 was analyzed using real-time RT-PCR on PBMC from 28 healthy individuals, 25 RRMS patients, and 20 PPMS patients. The data are normalized to the housekeeping gene, 18S rRNA, and are expressed as the mean relative expression ± SEM. *P < 0.05. C) CX3CR1 protein expression was quantified by flow cytometric analyses performed on PBMC from 19 RRMS patients and 19 healthy individuals. Data are shown as the mean protein expression ± SEM. *P < 0.05.

For the protein analysis we focused on patients with RRMS in whom we found reduced expression of CX₃CR1 positive lymphocytes (Fig. 1C ).

3. Reduced expression of CX₃CR1 on natural killer (NK) cells, but not on cytotoxic T cells from MS patients
Using multiparameter flow cytometric analysis, we investigated whether the expression of CX₃CR1 was down-regulated in all lymphocytes in MS patients or whether it was characteristic of a particular lymphocyte subpopulation. Analyzing the expression of CX₃CR1 on (NK) cells, CD4⁺ T cells and CD8⁺ cytotoxic T cells, we showed a reduced expression only on NK cells and not on CD8⁺ T cells. The receptor expression on CD4⁺ Tcells was almost undetectable in both patients and healthy controls.

4. Correlation of cytotoxicity with CX₃CR1 expression on NK cells
CX₃CR1-expressing CD8⁺ T lymphocytes show significantly greater cytotoxic activity than CX₃CR1-negative CD8⁺ T cells. To investigate whether CX₃CR1^high NK cells also show greater cytotoxic activity than CX₃CR1^negative/low, we isolated NK cells from PBMC of healthy individuals by negative selection and sorted them into CX₃CR1^high and CX₃CR1^negative/low fractions. By measuring the cytotoxicity of both populations against K562 using the calcein-acetyoxymethyl release assay, we demonstrated that CX₃CR1^high NK cells are in fact more cytotoxic than CX₃CR1^negative/low cells.

5. Correlation between clinical disease manifestations and CX₃CR1 expression
A detailed analysis of CX₃CR1 expression in RRMS patients and HD indicated that 72% of the healthy individuals presented a "normally" high frequency of CX₃CR1⁺ NK cells (shaded sector of the pie chart) while 28% of them showed low or undetectable levels of receptor-positive cells (hatched sector of the chart) (Fig. 2 A). The patient profile, however, was exactly the opposite, with 70% of the patients showing little or no receptor-positive NK cells, while the remaining 30% presented a frequency comparable with that of healthy individuals (Fig. 2A ). We observed that the majority of the 30% of patients who showed a "normal" proportion of receptor-positive NK cells shared common clinical characteristics and were in an active disease phase, whereas patients with a low frequency of receptor-positive NK cells were in a stable condition. To study this correlation with disease activity more closely, we extended the analysis of CX₃CR1 expression on NK cells to include additional healthy individuals as well as patients with acute relapses or MRI activity. It was confirmed that the majority of patients (92%) in a stable condition presented a very low frequency of CX₃CR1-expressing NK cells while the majority of patients (89%) suffering from acute relapses, or with gadolinium (Gd) enhancing lesion in MRI, showed an increased frequency of receptor-positive NK cells (Fig. 2B ). The frequency of CX₃CR1⁺ NK cells in patients with active disease was comparable with the frequency of those cells in healthy controls and four times higher than in stable patients (Fig. 2B ).

View larger version (41K): [in this window] [in a new window]   Figure 2. Expression of CX3CR1 on NK cells in RRMS patients and HD and in the disease course. A) The pie chart shows the frequency of NK cells expressing low levels of CX3CR1 (dark sector) vs. the frequency of NK cells expressing high levels of CX3CR1 (shaded sector). The diagram demonstrates that patients and HD present an inverse distribution of the NK cell subgroups: in contrast to the HD, 30% of the patients display a regular CX3CR1+ NK cell population, while 70% of these show an NK cell population with reduced CX3CR1 expression. B) Patients displaying a regular CX3CR1+ NK cell population are those with active disease (i.e., suffering from acute relapses or with gadolinium enhancing MR lesions) (hatched bar) while the patients with a stable disease course show decreased frequency of CX3CR1+ NK cells (dotted bar). The dark gray bar indicates the mean frequency of CX3CR1+ NK cells for all 22 patients together. The y-axis shows the frequency of CX3CR1-positive cells. Bars represent the mean expression of the receptor from 28 HD, 13 stable patients, and 9 patients with active disease, ± SEM. *P < 0.05, **P < 0.01.

CONCLUSIONS AND SIGNIFICANCE

We used large-scale gene expression analysis and quantitative real-time RT-PCR to identify marker genes for both RRMS and PPMS patients. Our findings emphasize the role of immune and cell cycle related genes in MS and, moreover, highlight the role of transmigration related mechanisms in the disease. One such gene involved in chemoattractant processes was that for the fractalkine receptor CX₃CR1. Here we show that PBMC from MS patients contain a decreased frequency of CX₃CR1-positive mononuclear cells when compared with that found in healthy individuals. We observed that this diminished expression exclusively affected NK cells, and not the cytotoxic T cell population, on both the RNA and protein levels. CX₃CR1⁺ NK cells express high levels of CD57, CD11b and CD11a and have numerous intracellular cytotoxic granules containing perforin and granzyme. Our data indicate that sorted CX₃CR1^negative/low are less cytotoxic than CX₃CR1^high NK cells. These cells therefore represent a cytotoxic effector population. The reduced frequency of CX₃CR1⁺ NK cells demonstrated in our study might imply the presence of a defective cytotoxic effector NK cell population in MS patients, which is in line with several reports of deficient NK cell activity in the disease. NK cells appear to be essential in regulating the immune response and in the development of autoimmune processes. Their immunoregulatory properties have been demonstrated in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), in which NK cells are reported to be involved in protection against autoimmunity.

The fact that an increased CX₃CR1 expression on CD8⁺ T cells has been found in patients with rheumatoid arthritis while patients with systemic lupus erythematosus and psoriasis show normal expression of the receptor on T lymphocytes and NK cells, respectively, indicates that a differential CX₃CR1 expression on lymphocytes is a phenomenon specific to MS and does not accompany autoimmune disorders in general.

Whereas the overall frequency of CX₃CR1-expressing NK cells was decreased in stable RRMS, the occurrence of CX₃CR1⁺ NK cells was in fact increased during relapses or active phases compared with their occurrence in healthy individuals. This was also confirmed by our microarray analysis, in which we observed that the only patient suffering a relapse after venipuncture showed an inverted/reversed regulation pattern of the chemokine receptor gene CX₃CR1. CX₃CR1 expression in patients, similar to the expression in HD, clearly correlates with acute relapses and/or with the presence of gadolinium enhancing magnetic resonance lesions. The precise role of NK cell subsets in MS patients has so far not been explained. Recently, it has been shown that NK cells involved in clinical remission are CD95-positive, produce type 2 cytokines, and are able to inhibit autoreactive cells. In contrast, CX₃CR1⁺ NK cells are known to produce type 1 cytokines. Thus the increase of CX₃CR1⁺ NK cells during relapses reported here may indicate a critical role of this proinflammatory subset of NK cells in disease exacerbation in addition to the more general immune regulatory capacity associated with these cells.

Our data highlight the role of innate immune mechanisms in MS pathology. The quantification of CX₃CR1⁺ NK cells represents a novel immunological parameter, not only for monitoring NK cell activity and facilitating MS diagnosis, but also as a reliable tool for monitoring disease course.

View larger version (34K): [in this window] [in a new window]   Figure 3. Using different methods to analyze gene and protein expression in MS patients vs. healthy individuals, we identified the expression of CX3CR1 on NK cells as a marker for MS patients and disease activity.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3832fje;

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