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Gene expression profiling of peripheral blood mononuclear leukocytes from psoriasis patients identifies new immune regulatory molecules

European Journal of Dermatology. Volume 15, Number 4, 251-7, July-August 2005, Investigative report

Summary

Author(s) : Dirk Koczan, Reinhard Guthke, Hans-Jürgen Thiesen, Saleh M Ibrahim, Günther Kundt, Helga Krentz, Gerd Gross, Manfred Kunz , Institute of Immunology, University of Rostock, Schillingallee 70, 18055 Rostock, Hans Knoell Institute for Natural Products Research, Beutenbergstr. 11a, 07745 Jena, Institute for Medical Informatics and Biometry, University of Rostock, Rembrandtstr. 16-17, 18057 Rostock, Department of Dermatology and Venereology, University of Rostock, Augustenstr. 80-84, 18055 Rostock, Germany..

Summary : In the present report gene expression profiling of peripheral blood mononuclear cells (PBMC) from psoriasis patients suffering from severe generalized disease was performed comparing diseased stage with cured stage. By this means, 18 genes were identified which showed differential expression. The most significant differences were found for IL-8, annexin A3, cycloxygenase-2 (COX-2), cell cycle regulator G0S2, and pre-B cell enhancing factor (PBEF), all of which showed upregulation in the diseased stage. Microarray data were confirmed by real-time RT-PCR. Further analyses using support vector machines identified three pairs of genes (IL-8 – CDKN1C/p57, cyclooxygenase-2 – NR1D2, and desmocollin-2 – CDKN1C/p57) which allowed an accuracy of disease stage prediction of 86%, based on gene expression patterns. Taken together, this is the first large-scale gene expression study of psoriasis PBMC identifying candidate genes that might contribute to psoriasis immunopathogenesis. The genes identified in the present report and the molecular mechanisms underlying their regulation might serve as future targets for therapeutic intervention in psoriasis.

Keywords : ANXA3, annexin A3, COX-2, cyclooxygenase-2, CDKN1C, cell cycle dependent kinase inhibitor 1C, DSC2, desmocollin-2, IL-8, interleukin-8, PBEF, pre-B cell enhancing factor

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ARTICLE

Auteur(s) :, Dirk Koczan¹, Reinhard Guthke², Hans-Jürgen Thiesen¹, Saleh M Ibrahim¹, Günther Kundt³, Helga Krentz³, Gerd Gross⁴, Manfred Kunz^4,*

¹Institute of Immunology, University of Rostock, Schillingallee 70, 18055 Rostock
²Hans Knoell Institute for Natural Products Research, Beutenbergstr. 11a, 07745 Jena
³Institute for Medical Informatics and Biometry, University of Rostock, Rembrandtstr. 16-17, 18057 Rostock
⁴Department of Dermatology and Venereology, University of Rostock, Augustenstr. 80-84, 18055 Rostock, Germany.

accepté le 5 Avril 2005

Psoriasis is an inflammatory skin disease of high incidence affecting about 2% of the population. It is regarded as a genetically inheritable disease [1, 2]. The typical clinical picture shows erythematous, scaly papules and plaques of variable size which may even extend into generalized erythroderma. In histopathology, a mixed inflammatory infiltrate is found, mainly consisting of lymphocytes and monocytes [3]. But there may also be an admixture of neutrophilic granulocytes.In the past years it has been shown that psoriasis skin lesions respond to immunosuppressive agents, such as macrolide immunosuppressants, cyclosporine and methotrexate [4-8]. PUVA bath therapy, another commonly used treatment modality in psoriasis, mainly acts on immune cells within the dermis and epidermis [9]. Thus, the role of activated lymphocytes and monocytes in the immunopathogenesis of psoriasis is now widely accepted. Moreover, evidence was provided that lymphocyte activation in the peripheral blood plays a central role in the pathogenesis of psoriasis [10]. In recent years, there have been discussions suggesting that psoriasis might even be regarded as an autoimmune disease [11-13]. Accordingly, systemically administered anti-lymphocyte and anti-TNF-α antibodies are successfully used for the treatment of psoriasis and have led to a real breakthrough in the treatment of psoriasis patients [14-19].A series of earlier reports analyzed gene expression in the microenvironment of psoriasis skin lesions [20-23]. These studies, on a limited number of genes, were extended by more recent large-scale gene expression studies which dramatically increased our knowledge about inflammatory mediators active in lesional psoriatic skin [24-26]. Among the genes identified in these studies were molecules of the S100 family of calcium binding proteins, chemokines like IL-8 and monocyte chemotactic protein-1, and cell signaling molecules like CD47 (integrin-associated signal transducer) [24, 25]. In the most extensive study analyzing 63,100 probe sets, a series of 131 differentially expressed so-called immune signaling genes were identified. These were divided into the major subgroups, interleukin-1 cluster, T-cell and dendritic cell activation, and chemokines [26]. Also, important transcription factors for cytokine regulation like STAT1 were identified [25, 26].However, the genes involved in the immunological activation of peripheral blood lymphocytes and monocytes have been poorly defined so far. In the present report, we performed a large-scale gene expression study for PBMC from psoriasis patients, comparing gene expression profiles in the diseased stage with that in the cured stage (after conventional dithranol treatment). Dithranol, also termed anthralin, is a classical compound used for external treatment of psoriasis [27]. For gene expression profiling, the oligonucleotide microarray technique was used for the parallel analysis of 12,000 genes. Overall 18 genes showed significant differential expression between both disease stages. Among these were a series of well-known inflammatory mediators such as IL-8 and cyclooxygenase-2, but also some recently identified immune regulatory molecules such as G0S2 and CDKN1C. Further analysis of differentially expressed genes by use of support vector machines identified three pairs of genes which allowed disease stage prediction based on gene expression patterns.

Materials and methods

Patients

Eleven patients suffering from severe generalized psoriasis who were administered to our outpatient clinic at the Department of Dermatology and Venereology, University of Rostock, were selected for mRNA expression profiling of PBMC. Patients included in this study displayed a skin involvement of more than 60% and a degree of severity of erythema, infiltration, and scaling of lesions of 3 or 4 (range 0-4) according to Psoriasis Area and Severity Index (PASI) [28]. None of these patients reported a recent infection or suffered from other unrelated severe diseases. Also, none of them suffered from psoriasis arthritis. Routine blood examinations excluded gross abnormalities. Patients were treated with increasing concentrations of dithranol until complete clearing of the skin lesions, which was achieved after 4-6 weeks. Blood samples were taken in the disease stage (immediately before treatment) and in the cured stage (8 days after the end of treatment and clearing of skin lesions).

Isolation of peripheral blood mononuclear cells (PBMC) and RNA extraction

Peripheral blood mononuclear cells (PMBC) were isolated from blood samples taken before treatment and after clearing of skin lesions. Leukocytes were separated using Ficoll density gradient centrifugation (Ficoll Hypaque, Sigma, Deisenhofen, Germany). This technique allows the separation of the lymphocyte/monocyte fraction (PBMC) from the granulocyte fraction. Cell smears were prepared and stained with hematoxylin and eosin in order to confirm the purity of the isolated cell population. Total RNA was isolated from PBMC preparations using the total RNeasy kit (Qiagen, Hilden, Germany).

Gene expression analysis of microarray data

RNA concentrations were determined spectrophotometrically at 260 nm and RNA probes were labelled according to the supplier’s instructions (Affymetrix, Santa Clara, CA, USA). Analysis of gene expression was carried out with Human Genome HGU95A microarrays (Affymetrix) interrogating 12,000 well-characterized human genes. Hybridization and washing of gene chips was done according to the suppliers’s instructions. Microarrays were analyzed by laser scanning (Gene Array™ Scanner, Hewlett-Packard) and the gene expression levels were calculated with the Microarray Suite™ 5.0 and Data Mining Tool™ 3.0 software (Affymetrix).

Real-time RT-PCR analysis (TaqMan™ assay)

Real time RT-PCR analyses (TaqMan 7700™; Applied Biosystems, Darmstadt, Germany) were performed to analyse gene expression of selected genes in larger series of psoriasis patients. These studies were performed for IL-8 (n = 32), annexin A3 (n = 23), G0S2 (n = 20), PBEF (n = 24), and COX-2 (n = 13). The numbers given in parentheses include the eleven patients already tested by microarray analysis. Gene expression analyses by real-time RT-PCR were carried out as described earlier [29]. Commercially available primer and probe sets were used (Assays-on-Demand; Applera Germany, Weiterstadt, Germany). The differences in gene expression between the diseased stage and cured stage were calculated by use of the ΔΔCt method [30]. Gene expression values were normalized to GAPDH expression.

Statistical analysis

Microarray data were subjected to paired Student’s t-test comparing gene expression in diseased stage and cured stage. In order to avoid false positive results, genes were excluded which showed upregulation in the cured stage and were represented with an absent call on the microarray at this stage, and genes that showed down-regulation in the cured stage and were represented with an absent call on the microarray at the diseased stage. Because peripheral blood leukocytes are a heterogenous population and enhanced or reduced gene expression might be restricted to particular populations or even sub-populations of cells a cut-off of at least 1.5-fold (instead of widely used 2-fold) up- or downregulation was used. Under these conditions, overall 18 genes (19 probe sets) were identified which showed significant differences in gene expression (p-value ≤ 0.05). Five of the genes were further validated by real-time RT-PCR analysis. Real-time RT-PCR data were subjected to paired Wilcoxon test. Bonferroni correction was applied and data with a p-value ≤ 0.05 after Bonferroni correction were regarded as statistically significant. Statistical analyses of microarray data were extended using a Support Vector Machine (SVM) algorithm with linear kernel (software may be downloaded from: http://www.eleceng.ohio-state.edu/~maj/osu_svm/) and cross-validation by leave-one-out method (own MATLAB-program). An accuracy of class prediction of 86% (three false positives or false negatives out of 22 samples tested) was regarded as significant.

Results

Microarray analysis of gene expression in PBMC from psoriasis patients

Overall 18 genes were identified which showed at least 1.5-fold up- or downregulation in gene expression comparing diseased stage with cured stage. Among these, 17 genes showed upregulation in the diseased stage. Only one gene showed upregulation after treatment in the cured stage (summarized in table 1( Table 1 )). The most striking upregulations in diseased stage were observed for IL-8 (3.8-fold). IL-8 exerts a large variety of different functions. It is particularly involved in lymphocyte/monocyte chemotaxis and activation. It is also a strong inducer of lymphocyte proliferation and cell cycle progression [31]. A further molecule with significant differences in gene expression was cyclooxygenase-2 (2.7-fold). It is a central enzyme in arachidonic acid metabolism leading to the generation of prostaglandins and thromboxanes [32]. Among various other functions prostaglandins are strong vasodilators, a major prerequisite for local tissue inflammation. G0S2 protein showed 2.6-fold upregulation. G0S2 is a member of the G0S family of G0/G1 switch genes. These molecules have been shown to be involved in lymphocyte cell cycle regulation [33, 34]. The precise role of pre-B cell enhancing factor (2.3-fold upregulation) has not been defined so far. Interestingly, however, this factor was initially identified in a cDNA library of activated lymphocytes derived from peripheral blood. It may be induced by strong inflammatory stimulators such as interleukin-1β and TNF-α [35]. CDKN1C was the only gene that showed upregulation after treatment with a mean fold of induction of 1.5. CDKN1C, also termed p57Kip2, is an inhibitor of cell cycle progression. These latter findings were suggestive for a role in lymphocycte/monocyte de-activation in the clinically cured stage of psoriasis patients.
Table 1 Description of differentially expressed genes in microarray analyses

Number	Affymetrix ID number	Gene bank accession number	Gene name	p-value	Mean of fold change
up-regulated in the cured stage
1.	1787_at	U22398	CDKN1C	0.0411	1.5960
up-regulated in diseased stage
1.	1369_s_at	M28130	IL8	0.0245	3.8297
2.	31792_at	M20560	ANXA3	0.0498	3.3007
3	35372_r_at	M17017	IL8	0.0050	3.1982
4.	1069_at	U04636	COX2	0.0322	2.7308
5.	38326_at	M69199	G0S2	0.0192	2.6035
6.	1372_at	M31165	TNFAIP6	0.0227	2.4362
7.	33849_at	U02020	PBEF	0.0065	2.3138
8.	1005_ at	X68277	DUSP1	0.0499	2.0280
9.	34319_at	AA131149	S100P	0.0257	1.9095
10.	2094_s_at	K00650	FOS	0.0189	1.8674
11.	34777_at	D14874	ADM	0.0479	1.6898
12.	35705_at	D16815	NR1D2	0.0232	1.6301
13.	31793_at	AL036554	DEFA3	0.0484	1.5344
14.	37185_at	Y00630	SERPINB2	0.0295	1.5226
15.	1915_s_at	V01512	FOS	0.0233	1.5207
16.	34435_at	AB008775	AQP9	0.0449	1.5184
17.	39302_at	X56807	DSC2	0.0142	1.5003
18.	37544_at	X64318	NFIL3	0.0012	1.5029

Validation of gene expression data from microarray analyses by real-time RT-PCR

In order to validate the data from the microarray analyses by an independent method real-time RT-PCR was performed for a larger series of patients. These analyses included the already tested patients by microarray analysis. Indeed, real-time RT-PCR data confirmed our findings from the microarray analyses. Even after Bonferroni correction four out of the five genes tested (IL-8, cyclooxygenase-2, annexin A3, and G0S2) showed statistically significant differences in gene expression. One gene, pre-B cell enhancing factor, showed a strong tendency for statistical significance (p = 0.07) after Bonferroni correction. Real-time RT-PCR data are summarized in table 2( Table 2 ). The differences observed in gene expression for G0S2 were even greater than those observed in microarray analyses. This might be due to an overall higher sensitivity of real-time RT-PCR (TaqMan^®). However, both techniques may not directly be compared because different nucleotide sequences for primers and probes and different amplification and signal detection techniques are used.
Table 2 Description of differentially expressed genes in real-time RT-PCR analyses

Number	Gene name	Median of fold change	p-value	p-value after Bonferroni correction	n
1.	IL8	1.7092	0.008	0.04	32
2.	G0S2	3.2713	0.0003	0.002	20
3.	PBEF	1.5542	0.014	0.07	24
4.	COX2	1.6175	0.008	0.04	13
5.	ANXA3	1.5548	0.003	0.015	23

Disease stage prediction by use of support vector machines

In an attempt to further optimize discrimination between both disease stages pattern recognition for gene expression values was performed using support vector machines together with cross validation (leave-one-out method). By this means three pairs of genes were identified with an accuracy of class prediction for disease stage of 86% ( (figure 1A-C) ). These pairs of genes consisted of IL-8–CDKN1C, cyclooxygenase-2–NR1D2, and desmocollin-2–CDKN1C. Further pairs generated by this method resulted in significantly less prediction accuracy. Together with CDKN1C, IL-8 was identified as a major discriminator between diseased and cured stage. Thus, both molecules might be of importance for lymphocyte/monocyte activation and cell cycle regulation in psoriasis. Interestingly, two pairs of genes contained the cell cycle dependent kinase inhibitor 1C. Indeed, it was recently shown that CDKN1C acts as a potent inhibitor of T lymphocyte activation [36]. The gene NR1D2 is an orphan nuclear receptor presumably involved in gene regulation. However, its precise role is unknown at present. Desmocollin-2 is a well-known cell-cell adhesion molecule of the cadherin family. It mediates epithelial cell-cell contacts. Its role in inflammatory cells remains to be defined so far. Taken together, inflammatory activation and cell cycle regulation mediated by IL-8 and CDKN1C appear to be a central mechanism in the control of inflammation in psoriasis patients.

Discussion

Gene expression profiles of psoriasis PBMC identified 18 genes overall which showed significant up- or downregulation in the diseased stage compared with the cured stage. Microarray data were further validated by real-time RT-PCR. Among the genes with enhanced expression in the diseased stage were well-known inflammatory mediators like IL-8 and cyclooxygenase-2, and molecules that had only recently been described to be involved in lymphocyte/monocyte activation, like pre-B cell enhancing factor, annexin A3, G0S2, and CDKN1C. Cross validation using support vector machines was performed for differentially expressed genes from microarray analyses. By this means, three pairs of genes were identified that allowed a disease stage prediction with an accuracy of 86%.

In the study presented, interesting candidate molecules for the immunopathogenesis of psoriasis were identified. Among these, IL-8 might play an outstanding role, since this molecule showed the greatest difference in gene expression between diseased stage and cured stage. IL-8 has various immune regulatory functions and its expression had been shown to be strongly upregulated in psoriasis skin lesions [21, 22, 37-39]. Besides its role as a strong chemoattractant for inflammatory cells, it directly induces lymphocyte and monocyte activation. The particular role of IL-8 in psoriasis skin lesions was further emphasized by two recently published large-scale gene expression studies using microarray technique [24, 25]. A third, also recently published microarray study analyzing gene expression in psoriasis identified both upregulated IL-8 and upregulation of one of its two corresponding receptors, namely IL-8Rβ, in psoriasis skin lesions as compared with normal control skin [26].

In accordance with the data presented, it was shown earlier that peripheral blood monocytes from psoriasis patients display enhanced IL-8 expression [40, 41]. A further study showed that both corresponding receptors for IL-8 were upregulated on PBMC from psoriasis patients [42]. Thus, IL-8 may act as an activator for lymphocytes/monocytes and induce lymphocyte/monocyte proliferation and differentiation in the peripheral blood compartment. Moreover, it has been shown that IL-8 may directly activate monocytes and promote adhesion of monocytes to endothelial cells which might be regarded as a major prerequisite for inflammatory tissue invasion [43]. The same mechanism might activate lymphocytes, however, experimental data are lacking.

In a recent study, the efficacy of oral pimecrolimus therapy for psoriasis was demonstrated, resulting in 80% improvement of skin lesions after 4 weeks of treatment [44]. IL-8 expression in whole blood samples from these patients did not change significantly when measured after 14 days of treatment. This lack of IL-8 response to treatment might be explained by that fact that only a 30% improvement of skin lesions had been achieved at that time point. In contrast, a total clearing of skin lesions was achieved in the study presented which might be a prerequisite for IL-8 downregulation. However, it cannot be ruled out that the observed effects on IL-8 gene expression may at least in part be due to direct effects of dithranol. Interestingly, in the microarray analyses conducted by Rappersberger and co-workers analysing the expression profile of 7,129 genes, significant changes in gene expression were observed for molecules involved in prostaglandin metabolism and thromboxane A2 receptor binding [44].

Relative treatment resistance of psoriasis skin lesions to anti-IL-8 therapy (ABX-IL8) may be somewhat contradictory to the presented findings [45]. However, this may be explained by a redundancy in immune regulating molecules in psoriasis. Possible candidates for redundant immune activation compensating for targeted downregulation of IL-8 might be monocyte chemotactic protein 1 or monokine induced by interferon-γ [20-22]. However, further investigations are necessary to address this question.

Two genes involved in lymphocyte cell cycle regulation, G0S2 and CDKN1C, were identified in the study presented. These findings were suggestive for a tightly controlled cell cycle regulation of lymphocytes in psoriasis. Little is known about the precise role of cell cycle molecules of the G0/G1 switch (G0S) family. G0S2 expression had been shown to be induced in cultured peripheral blood mononuclear cells after stimulation by lectin, or by a combination of the phorbolester TPA and calcium ionophore ionomycin [33, 46]. Moreover, cyclosporine, a well-known immunosuppressive drug inhibits G0S2 expression. Together, these data are suggestive for a role of G0S2 in lymphocyte activation. The second molecule identified as being involved in cell cycle regulation, CDKN1C, was the only gene in our study which showed significant up-regulation in the cured stage. Indeed, CDKN1C, which has also been termed p57Kip2, is a cell cycle regulator which exerts anti-proliferative effects. Its particular role for lymphocyte cell cycle regulation has been described very recently [36]. In this latter study the CDKN1C gene product associates with different cyclins. It has been demonstrated that overexpression of CDKN1C in Jurkat cells resulted in inhibition of cyclin E- and cyclin A-associated cdk2 kinase activity and a decrease in amounts of cyclin A. The net result of these interactions was an anti-proliferative effect on lymphocytes. These experimental findings are in accordance with the detection of up-regulated CDKN1C expression in the cured stage of psoriasis patients in our study. Interestingly, CDKN1C was one of the few genes which showed reduced expression in diseased versus normal skin in one of the recently mentioned published microarray studies on psoriasis [26].

Common findings in psoriasis histopathology are widened, so-called tortuous capillaries in the dermal papillae. In the present report, we identified cyclooxygenase-2 as a candidate gene which might help to explain these histopathological findings. Cyclooxygenase-2 produces prostaglandins and thromboxanes from the ground substance arachidonic acid [32]. Prostaglandins are well-known and strong vasodilators. Thromboxanes on the other hand are central molecules in the blood coagulation system and exert pro-coagulant functions. The induction of these molecules by cyclooxygenase-2 might induce widening of capillaries and promote tissue inflammation in concert with other local factors. Interestingly, enhanced cyclooxygenase-2 expression had been described in platelets of psoriasis patients [47].

A further molecule which showed upregulation in active disease was annexin A3. Annexins are membrane-bound phospholipds highly expressed in lipid rafts of the outer cell membrane [48]. Lipid rafts are central areas involved in lymphocyte-dendritic cell interaction promoting lymphocyte activation [49, 50]. Besides its role in cell adhesion, cell signaling and membrane integrity, evidence has been provided that annexin A3 is involved in monocyte differentiation [51]. Furthermore, it had been shown that annexins interact with molecules of the S100 family of calcium binding molecules [48]. Members of this family are suggested to play an important role in psoriasis pathogenesis, a fact that was emphasized by three large-scale gene expression studies on psoriatic skin [24-26]. All the studies mentioned showed upregulation of members of the S100 family in psoriatic lesional skin. Among these were S100A7-A9, S100A11, and S100A12. In earlier reports, S100A7 had been termed psoriasin, referring to its suggested pathogenic role in this disease [52].

The data presented demonstrated upregulation of PBEF, a B cell stimulation factor, in the diseased stage. Little is known about the role of B cells in psoriasis pathogenesis. However, in one earlier study, B cells were detected in psoriatic lesional skin [53]. Even more important, it has recently been shown that PBEF expression was induced in cultured monocytes after LPS stimulation [54]. Enhanced PBEF expression has also been found in one of the mentioned microarray studies in psoriatic lesional skin [26]. Thus, overall, three of the molecules identified in PBMC in the present study, IL-8, CDKN1C, and PBEF have been described in at least one other of the three large-scale gene expression studies mentioned of psoriasis skin biopsies.

To further substantiate the findings presented, microarray data were analyzed by support vector machines. Support vector machines are self-learning computer programs which have been used recently for the analysis of gene expression microarray data [55]. These analyses allow class (disease stage) prediction of investigated samples based on the analysis of gene expression profiles of multiple genes. Here we show that 3 pairs of genes allowed a disease stage prediction with an accuracy of 86%. It might be concluded that genes which form pairs or even larger groups of genes in these analyses are functionally related or may even interact. One of these pairs contained IL-8 and CDKN1C. Interestingly, both showed opposite regulation. From a functional point of view, enhanced IL-8 expression may downregulate CDKN1C expression or vice versa. This question may not be answered by the data presented. However, these findings should stimulate further functional investigations which might have an important impact on future therapy. Two further pairs of genes identified by support vector analysis allowed a discrimination of both disease stages with the same accuracy of prediction. Again CDKN1C was one partner, and desmocollin-2 (DSC2) the other. Desmocollin-2 is a well-known cell-cell adhesion molecule. It is a member of the cadherin family of cell adhesion molecules involved in desmosome formation, tight cell-cell contacts. These play a particular role in epidermal desmosomal junctions. At present, nothing is known about its role in inflammatory cell interactions or PBMC endothelial cell interaction.

Taken together, this is the first large-scale gene expression study of psoriasis peripheral blood mononuclear cells. Interesting target molecules were identified which might be of importance for the immunopathogenesis of this disease. The significance of these findings was further supported by the fact that the pairs of particular genes allowed disease stage prediction with high accuracy. The molecules identified in the presented study might serve as new therapeutic targets for innovative treatment modalities for this chronic inflammatory disease or might be used as marker molecules for disease activity.

Declaration: Local Ethical Committee approval was received for the studies and the informed consent of all participating subjects was obtained.

Acknowledgments

We thank R. Waterstradt and I. Todt for excellent technical assistance.

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