Human Molecular Genetics, 2001, Vol. 10, No. 17 1793-1805
© 2001 Oxford University Press
Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies
1Department of Internal Medicine and 2Departments of Genetics and Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA, 3Naval Medical Research Center, Georgetown University, Kensington, MD 20895, USA and 4Department of Internal Medicine, Division of Dermatology, Baylor University Medical Center, Dallas, TX 75246, USA
Received April 12, 2001; Revised and Accepted June 24, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Approximately 2% of the Caucasian population is affected by psoriasis (PS); a chronic inflammatory skin disease triggered by both genetic and environmental risk factors. In addition to a major contribution from the HLA class I region, PS susceptibility loci have been mapped to a number of regions including 1q21, 3q21, 4qter, 14q31–q32, 17q24–q25, 19p13.3 and 20p. Some of these overlap with loci implicated in other autoimmune/inflammatory diseases. Global gene expression studies are beginning to provide insights into the etiology of these and other complex diseases. We used Affymetrix oligonucleotide arrays comprising approximately 12 000 known genes to initiate a more comprehensive analysis of the transcriptional changes that occur in involved and uninvolved skin of 15 psoriatic patients versus six normal controls. Expression levels of the transcripts detected on the arrays were first used to determine the relationship of samples to each other using hierarchical clustering. This analysis clearly differentiated involved psoriatic skin from uninvolved and normal skin. Clusters of differentially expressed genes with similar expression patterns in the same samples were then identified. Six out of 32 clusters contained a total of 177 transcripts that were differentially expressed in involved psoriatic skin versus normal skin. These differences were independent of the gender, age, skin site and HLA class I status of the patient. Ten of the 177 genes were also differentially expressed in uninvolved skin, and several mapped to regions previously shown to harbor psoriasis susceptibility loci.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Psoriasis (PS) is a common inflammatory skin disease affecting
2% of individuals from the northern European population (1). The three cardinal features of psoriatic lesions are erythema, induration and scaling. Areas of involvement are usually well demarcated and distributed in a characteristically symmetrical manner (2). An association with psoriatic arthritis is seen in 6–10% of cases (3). In young patients it is frequently triggered by infection with group A streptococci (4), and can also occur with human immunodeficiency virus (HIV) infection (5,6). As with most autoimmune diseases, there is a strong contribution of HLA alleles, particularly Cw6 and B57 (7–12). PS can also be independent of HLA, and genetic linkage studies of multiply affected families have mapped susceptibility loci to chromosomes 1q21, 3q21, 4q, 14q31, 17q24–q25 and 19p13 (1,13–17) (unpublished data). Epistasis with HLA has been described for some of these loci (e.g. 1q21) (18). Genome scans of large numbers of affected sibling pairs and nuclear families have confirmed linkages to 1q21, 14q31 and 17q24.3 and have suggested the existence of additional loci on chromosomes 1p, 16q and 20p (12,19,20) (unpublished data). Some PS susceptibility loci coincide with loci identified in genome-wide scans of other autoimmune/inflammatory diseases including chromosomes 1q21 [atopic dermatitis (AD)] (21), 3q21 [rheumatoid arthritis (RA) and AD] (22,23), 14q31 [Grave’s disease (GD), insulin dependent diabetes mellitus (IDDM)] (24,25), 16p [irritable bowel disease (IBD)] (26) and 17q24.3 (RA) (27). This is in agreement with the hypothesis that in some cases, clinically distinct autoimmune diseases may be controlled by a common set of susceptibility genes (28) (reviewed in ref. 29). Overlap of some of these regions with diseases like childhood AD (21), suggests that inflammatory diseases in general are due to an overlapping set of susceptibility loci.
A number of changes in gene and/or protein expression have been described previously in PS and some overlap with other inflammatory diseases. IL-1 and TNF-
are up-regulated in psoriasis and activate several cellular signaling pathways including the NF-
B pathway. Genes regulated by NF-B include those central to cutaneous inflammation such as E-selectin, chemokines, cytokines, defensins, intercellular adhesion molecule 1 (ICAM1) and vascular adhesion molecule 1 (VCAM1) (30). Psoriatic keratinocytes also elaborate HLA-DR (31), 
-interferon (IFN-), (32,33); CD36 (OKM5) (33), -interferon-induced protein 10 (-IP10), IL-1 (33) and TGF- (34,35). These and other studies on small groups of genes (36) have provided valuable initial insights into the molecular basis of psoriasis. The changes observed in these and other studies provide important internal controls for more global gene expression analyses such as the one described in this study.
We used Affymetrix oligonucleotide arrays comprising approximately 12 000 known genes to initiate a more comprehensive analysis of the transcriptional changes that occur in involved and uninvolved skin of psoriatic patients versus normal controls. Distances between samples, calculated on the basis of the expression levels of all genes detected on the arrays, clearly differentiated involved skin from uninvolved skin and normal samples. K-means clustering was then used to search for transcripts with similar expression levels in samples and resulted in the identification of 177 transcripts with altered expression in psoriasis. 161 of these were upregulated and less than one-fifth of these have been described previously in psoriasis. Ten were also up-regulated in uninvolved skin. Several of these mapped to regions previously linked to PS susceptibility and/or other autoimmune diseases.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Samples used in the study
Six millimeter punch biopsies were obtained from the skin (involved and uninvolved) of 15 unrelated Caucasian PS patients. This included matched pairs of uninvolved and involved skin from 12 patients, involved skin only from two patients and uninvolved skin only from one patient. Biopsies were also obtained from six unrelated normal Caucasian controls. A description of patients and the site from which the biopsies were obtained is provided in Table 1. DNA and RNA were obtained from each biopsy. RNA samples were used for the expression studies. DNA was used to determine the HLA class I status of each patient. Patients were genotyped for HLA-A, -B and -C alleles with routine PCR amplifications and hybridization with sequence-specific oligonucleotide probes.
|
RNA was prepared from all biopsies and gave average yields of 50–90 µg total RNA from the involved samples, 10–30 µg from the uninvolved samples and 10–17 µg from the normal samples. Ten to fifteen micrograms was used to prepare biotinylated cRNA and an aliquot (approximately one-fifth) was hybridized separately to Affymetrix U95A arrays that contain probes for approximately 12 000 known transcripts. Following hybridization and washing, GENECHIP software was used to convert the image intensities of each set of gene probes on the array to an ‘average difference’ value (referred to here as ‘expression level’). After negative or zero average difference calls were removed, a total of 7129 transcripts remained. These genes were analyzed in the 15 involved skin samples, 11 uninvolved skin samples and six normal controls.
Relationship between samples
Cluster analysis (37) is currently the most widely used statistical technique applied to large-scale gene expression data. One approach is to determine the relationship between samples and to illustrate this with a dendrogram (clustering tree). This requires hierarchical clustering; an approach used previously by Eisen et al. (38) who applied pairwise average-linkage cluster analysis to gene expression data. The expression profiles of each sample are compared with every other sample, and a distance measurement is assigned to each pair. We had 32 samples so there were 32 x 32 distances that were computed on the basis of the expression levels of all 7129 genes. The two samples with the closest distance are first merged and connected by branches with lengths representing their distance. The average of the standardized expression levels of the two samples (average-linkage) is used to determine their relationship with the remaining samples. The next pair of samples with the smallest distance is chosen to merge, and the process is repeated until all samples are merged.
The dendogram shown in Figure 1 illustrates the relationships between all 32 samples used in this study on the basis of expression levels of all 7129 genes. The y-axis in Figure 1 shows the distances at which clusters were joined. For example, samples PS 22 and PS 28 were joined first at a distance of 75, indicating these two samples have the most similar gene expression profiles among the 32 samples. These two samples were next clustered with sample PS 20 at a distance of ~90, based on the average distance of PS 22 to PS 20, and PS 28 to PS 20. It can be seen in Figure 1 that there is a strong separation of the involved psoriatic samples from uninvolved and normal samples. All 15 involved psoriatic skin samples were merged with distances less than about 140, and included only one uninvolved sample (NL 24). This involved psoriatic skin cluster was then merged with the remaining uninvolved and normal samples at or >140.
|
This separation of involved from uninvolved and normal skin samples implies the existence of some major gene expression differences that result in the differentiation of these two groups of samples. This separation is independent of the HLA class Cw*0602 status of the patients since the positive and negative patients did not fall into separate branches of the tree.
It was not possible to determine if the distances between the samples, and thus the separation between them, was significant. Cluster analysis must be viewed as a data reduction technique, and since no robust probability distributions exist for dendrograms, it is not possible to calculate statistical P-values for these results. In addition, an appropriate alternative hypothesis is difficult to specify.
Gene clustering
In order to identify genes that were differentially expressed in our samples we performed K-means clustering. This identifies clusters of genes with similar expression patterns in individual samples, taking all samples into account. Inspection of expression levels in individual samples (with scatterplots for example) will identify those gene clusters with specific patterns of expression in samples or groups of samples. One can select the number of clusters into which transcripts must fall, and we placed genes into 2–32 clusters. Thirty-two clusters were sufficient to identify several discrete groups of genes that had different expression levels in involved versus normal samples.
Visual inspection of scatterplots of these 32 clusters revealed six clusters of genes where expression levels in involved psoriatic skin differed from that in uninvolved and normal skin. The total number of independent genes in these six clusters were 177. These six clusters are discussed in detail below. Even with this small number of clusters, some genes with similar expression levels were split into two clusters (e.g. clusters 3 and 7, and clusters 26 and 29, see below). These were merged into single clusters. Other clusters did not differentiate involved skin from normal skin, or uninvolved skin from normal skin. A complete list of genes in clusters that differentiated involved skin from normal skin can be found on our web site (http://hg.wustl.edu).
Highly up-regulated genes
Clusters 3 and 7 contained genes with very high expression levels in involved psoriatic skin. The nine genes in cluster 3 and the 11 genes in cluster 7 correspond to 17 independent transcripts since oligonucleotide probe sets for several genes (e.g. keratin 16) were represented more than once on the array. Since the patterns of expression changes of the genes in these two clusters were so similar, they were grouped together into a single cluster. A description of the genes in this cluster (gene name, GenBank accession no., Unigene cluster no., chromosomal localization) is shown in Table 2. The overall expression levels of these 17 genes in individual samples are illustrated in the scatterplot in Figure 2. In this figure, the expression level is shown on the y-axis and the individual samples are shown on the x-axis. In order to compare gene expression levels of these 17 genes in normal, uninvolved and involved skin, samples have been arranged on the y-axis in that order. It can be seen that the expression levels of genes in this cluster rose to >80 000 in involved skin.
|
|
A comparison of fold-changes of involved versus normal skin, and uninvolved versus normal skin for each of these genes indicated a substantial amount of variability (3-fold for cystatin A and S100A2 versus 50-fold for SERPINB4).
Most of the genes in this cluster have been previously reported to be up-regulated in keratinocytes of involved psoriatic skin (referenced in Table 2). These genes provide a valuable internal control set that validates our observations. Within this set of up-regulated/induced genes we also identified a gene whose profound up-regulation in psoriasis has not been reported before. This gene encodes interferon -inducible protein 27 (IFI27) which maps chromosome 14q32. It was one of four transcripts within this cluster that was also up-regulated in uninvolved skin.
Other differentially expressed transcripts
Other up-regulated transcripts which clearly differentiated involved psoriatic skin from uninvolved and normal skin were found in clusters 11 (27 genes), 26 (42 genes) and 29 (83 genes) (Fig. 3A and B and Tables 3 and 4). As with clusters 3 and 7, some genes were found in more than one cluster because they were represented by several probes on the U95A array. Since the differences in the expression levels of genes in clusters 26 and 29 were similar they were also grouped together, resulting in a non-redundant set of 120 genes. Alterations in steady-state mRNA levels of most of the genes in clusters 11, 26 and 29 in PS have not been described previously.
|
|
|
A comparison of mean expression levels of these transcripts in involved and normal skin revealed substantial differences in fold-changes. The transcript with the greatest fold-change was transcobalamin I (vitamin B12 binding protein), a neutrophil granule protein that was up-regulated 93-fold in involved versus normal skin. Transcripts up-regulated in both involved and uninvolved skin were CD47, IL8, ECGF1, SPRR2C and STAF50.
There were 16 transcripts found in cluster 19 that were down-regulated in involved psoriatic skin compared with uninvolved and normal skin (Figure 3C and Table 4).
Reclustering of samples with a select set of differentially expressed genes
We reclustered samples with only the 177 differentially expressed genes identified by K-means clustering as being differentially expressed in involved versus normal skin. The resulting hierarchical average linkage tree is shown in Figure 4. The separation between involved and uninvolved/normal samples is even more pronounced than in Figure 2. This validates the genes identified by K-means clustering as contributing to the separation of involved versus uninvolved and normal samples.
|
Transcripts of low abundance
K-means clustering is a conservative approach of identifying subgroups of genes on the basis of expression profiles. Comparing the means of expression levels of involved versus normal skin revealed 454 genes with average fold changes of two or more. Many of these are likely to be bona fide differentially expressed transcripts that were found in clusters that did not differentiate involved from normal skin with K-means clustering.
A number of genes, previously described to be altered in expression in psoriatic skin, were not found within the set of 177 differentially expressed genes. These included IL1, IL2, IL6, IL8R, IL12, CD3, CD4, CD8, CD25 (high-affinity IL2 receptor), CD28, CD36, CD40L, CD69, CD86 (B7-2), CD122, IFN-, SCYB10 and TGF- (33,36,39–43). Many of these transcripts were found within two large clusters of genes with relatively low expression levels (cluster 6 that contains 2537 genes and cluster 13 that contains 1622 genes). Upon closer inspection it was seen that some transcripts were present at different levels in involved versus normal skin (for example, IL8R which was up-regulated 4-fold on average in involved versus normal skin). However, many transcripts did not even exhibit fold changes when involved and normal skin were compared. In contrast to uninvolved skin, involved skin contains a significant number of inflammatory cells that elaborate a number of the genes listed above. Failure to see differential expression of these genes in involved versus uninvolved skin needs to be examined further. Probes for some of the genes listed above were not found on the U95A array (IL2, IL12, IL1B, CD122, CD25, CD28, CD40 and EGFR).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
This is currently the largest study of gene expression changes in psoriatic skin. We identified a total of 177 genes that differed in expression in involved versus normal skin. We were able to find previous reports for a role of 32 of these genes (18%) in psoriasis. These previously described genes served as good internal controls for our study. Alterations in the expression of the remaining 145 genes have not been described previously in psoriasis. 161 transcripts were induced de novo or were up-regulated in involved skin and only 16 genes were detectably down-regulated. Novel genes within each cluster reflected the proliferative and inflammatory characteristics of psoriatic skin.
Reliable expression array data is clearly dependent upon conducting duplicate hybridizations and it is estimated that the error rate for an observed 2-fold change in Affymetrix expression data is 1% (44). Duplicate hybridizations reduce the chance of finding the same 2-fold change to 0.01%. Our strategy in designing this study was to search for consistency across a relatively large number of different samples, rather than to run duplicate hybridizations on a much smaller number of samples. By employing this strategy we ran the risk that small (~2-fold) changes within a given sample might be spurious, but that consistent changes across the entire set of samples would have a very high significance level.
Gene clustering
K-means clustering searches for patterns in large data sets, and was used to place transcripts into clusters with similar expression levels in individual samples. When we analyzed our data we identified six clusters of genes (clusters 3, 7, 11, 19, 26, 29) with expression levels that differentiated involved psoriatic samples from normal samples. Visual inspection of scatterplots of these clusters indicated that in general the expression levels of these transcripts in uninvolved skin was similar to that seen in normal skin. The skin biopsies used for this study came from very different regions of the body (e.g. leg, arm, abdomen, foot, back) and were from patients of different ages and of both sexes. Some of the other clusters of genes that do not distinguish involved skin from uninvolved and normal skin may reflect heterogeneity in gene expression in the skin due to these or other variables. The changes in the 177 genes within these clusters are therefore likely to point to changes with real relevance to disease.
Clusters 3 and 7 contained 17 genes that were highly up-regulated or induced and the differential expression of thirteen of these genes in psoriasis has been described elsewhere. A number of genes in these clusters (e.g. K6, K16, K17 and elafin) are not expressed in normal skin, and are characteristic of hyperproliferative keratinocytes (45). Many of the genes in these clusters have been shown to be induced in response to TNF- stimulation of keratinocytes (S100A8, S100A9, cystatin A, SPRR1B, SPRK, elafin) (45,46). The TNF- induction of elafin has been shown to be via a p38 MAP kinase-dependent pathway (45). IFI27 has not been described before in the context of this disease. It was previously shown to be up-regulated in 50% of breast carcinomas (47). Its role in the pathogenesis of PS is being investigated further. The possibility that these genes are all up-regulated due to the presence of a specific regulatory element is being investigated.
The majority of the changes in clusters 11, 19, 26 and 29 have not been described previously in psoriasis. These included an increase in the expression levels of the STAT1 and STAT3 genes, and alterations in the levels of large numbers of cysteine and serine proteinases (e.g. members of the kallikrein family), proteinase inhibitors (e.g. members of the SERPINB family) and components of proteasomes. One role of proteasomes is to generate peptides from intracellular endogenous and viral proteins for presentation by MHC class I molecules. -interferon has been shown to alter the expression levels of genes encoding proteasome subunits (48,49). It has been hypothesized that alterations in the catalytic specificity of proteasomes is due to substitution of one subunit for another which could determine which antigenic peptides are presented by the cell (49,50). This is in keeping with the HLA Class I association with PS and the presentation of an antigenic peptide derived from an as yet unidentified protein.
We were interested to note that within clusters the average fold-changes of transcripts varied. This was particularly pronounced in cluster 3/7 which contained the most highly up-regulated genes. For example, SERPINB4 was up-regulated 50-fold compared with normal skin, interferon -inducible protein and psoriasin were up-regulated ~12-fold, and cystatin A was up-regulated only 3-fold. Several genes in this cluster were also up-regulated in uninvolved skin, suggesting a role in psoriasis susceptibility.
Transcobalamin 1 up-regulation
The transcript exhibiting the greatest average fold change in expression was transcobalamin 1 (vitamin B12 binding protein) (cluster 26). It was up-regulated 93-fold on average when involved and normal skin were compared. It was not detectably up-regulated in uninvolved skin. It is found in the secondary granules of mature neutrophils. Its role in psoriasis has not been emphasized, although levels of TCN1 protein are correlated with the severity of intestinal inflammation in Crohn’s disease and RA (51–53). Elevation of TCN1 has been thought to be responsible for the elevation of vitamin B12 in a number of chronic myeloproliferative disorders (54). Defensins are also granule proteins of myeloid cells and ß-defensin is also up-regulated in involved skin, suggesting an important role of neutrophil granule proteins in psoriasis.
Genetic risk factors
The changes in gene expression of the 177 genes described here were also independent of known genetic factors such as the presence of the HLA class I associated allele, HLA-Cw*0602. Approximately half of our patients harbored at least one HLA-Cw*0602 allele, but the genes described here did not differentiate them from the HLA-Cw*0602 negative patients. Interestingly, one uninvolved skin sample (NL24) clustered with the involved skin samples in the hierarchical average linkage tree suggesting more similarity in gene expression of this sample with involved skin than uninvolved skin. When scatterplots of clusters 3 and 7 were evaluated, several uninvolved skin samples (NL16, 18 and 24) also showed expression in the range of involved skin samples. These samples were from clearly uninvolved areas of the skin of these patients. Interestingly, all of these patients were male and HLA-Cw*0602 negative, and two reported a family history of psoriasis.5
|
Chromosomal locations
A number of genes that were highly expressed in involved skin mapped to psoriasis susceptibility loci. These included genes from the epidermal differentiation complex (EDC) that map to chromosome 1q21, cystatin (3q21) and
-interferon inducible protein p27 (14q31). In the case of chromosome 1q21, a number of genes from the EDC were up-regulated. The EDC is involved in the calcium-dependent, terminal differentiation of stratified squamous epithelia. It harbors approximately 37 genes (55,56) including six encoding structural proteins of epidermal cornification (the cornifins or SPRRs), 13 S100 calcium-binding proteins, filaggrin, involucrin, trichohyalin and loricrin. The S100A2, S100A7, S100A8, S100A9, SPRR1B and SPRR2A genes were within the group of highly up-regulated genes in clusters 3/7. Two additional genes from the EDC were found in other clusters of up-regulated transcripts (involucrin, cluster 11; SPRR2C, cluster 29). The remaining genes from this complex were not detectably up-regulated on the U95A array. Three of these genes were also up-regulated in uninvolved skin (psoriasin or S100A7, SPRR2C and SPRR1B). Recent association studies localize the 1q21 linked/associated psoriasis locus to within the EDC (57). Up-regulation of genes within this cluster is reminiscent of the cis-acting regulatory elements (locus control regions) affecting the developmental regulation of multiple genes (58). One hypothesis accounting for the 1q21 linkage is a genetic variant affecting expression of a number of genes within the EDC.
In the case of 14q31–q32, we first observed some evidence of linkage to this region following a genome-wide scan of multiply affected families (1) (unpublished data). A peak NPL score of 8.96 was obtained with D14S617 (105.53 cM) in one large multiply affected family with psoriasis. A follow-up study on 250 nuclear families provided additional moderate evidence for linkage to this region (NPL = 1.29, P = 0.08) at D14S81 (108.22 cM) (unpublished data). Recent studies on a large cohort of PS families from the United Kingdom provided further evidence for linkage of psoriasis to this region (20). This region may harbor a susceptibility locus for autoimmune diseases in general since IDDM11 and GD have been mapped to this region as well (24,25). The polymorphic microsatellites that are closest to the gene encoding interferon, -inducible protein 27 are also those providing peak LOD scores in the PS and GD studies. This gene was up-regulated in involved and uninvolved psoriatic skin. Variants within this gene and its regulatory sequences are being investigated for susceptibility to psoriasis and for autoimmunity in general.
One might argue that the mapping of the highly up-regulated genes to susceptibility loci is due to the co-incidental mapping of an up-regulated gene to a large genomic region. However, this would be surprising in all cases since the overlapping regions were small and usually <5 cM (in a genome of 3000 cM). Our observation parallels a recent study on ulcerative colitis (UC) and CD where the map locations of a number of genes or gene clusters with differential expression in IBD lie within IBD susceptibility regions on chromosomes 12q13.2–q24.1, 19p13, 7q21.1 and 6p (59). However, in our study it was only up-regulated transcripts that mapped to PS susceptibility loci. One would expect that if a genetic defect resulted in an altered transcript level, it would be seen in uninvolved as well as involved skin. In fact, some of the up-regulated genes in these linked regions also exhibited differences in uninvolved versus normal skin (1q21 and 14q31). In other cases the alteration was only detected in involved skin (3q21), suggesting that the overlap might be co-incidental.
The identification of susceptibility loci for autoimmune diseases
The identification of genetic risk factors for complex diseases presents a challenge because of the moderate effects these factors may have on risk. An additional complexity is genetic heterogeneity. The possibility that gene expression studies may provide insights into novel biochemical pathways, and even possibly candidate genes for inflammatory diseases, would greatly speed up the identification of genetic risk factors for these complex diseases. These studies may identify common autoimmunity loci as well as disease-specific loci. Aberrant biochemical pathways leading to psoriasis may in turn harbor proteins that are amenable to novel therapies that lack the side effects associated with current treatments.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Samples used in study
Six millimeter punch biopsies were obtained from involved and uninvolved skin of patients and controls. The anesthesia used was 1% lidocaine with 1/100000 epinephrine, with 1–5 min between injection and biopsy. All patients had stable, chronic, plaque PS. Presence or absence of PS was determined in all cases from the patient’s medical history and clinical evaluation. Informed consent was obtained from all individuals subjected to skin biopsies. Protocols for obtaining patient biopsies had been approved by both Baylor Hospital in Dallas and Washington University Institutional Review Boards for the protection of human subjects. All patients were Caucasian. Table 1 provides additional information on patients (gender and family history).
Target preparation
Total RNA was prepared from all samples, amplified and labeled with biotin following the procedure described elsewhere (60).
Probe arrays
The arrays were synthesized using light-directed combinatorial chemistry as described elsewhere (61). The microarrays used for this study were U95A GeneChip® probe arrays (Affymetrix Inc., Santa Clara) that contain probe sets representing approximately 12 000 genes.
Fragmentation, array hybridization and scanning
The labeled target was fragmented, and hybridized to probe arrays according to (62). The probe arrays were then washed, stained and scanned (62).
Analysis
GeneChip® 3.2 software (Affymetrix Inc.) was used to scan the images, convert intensities to a numerical format and obtain an average difference value for each probe on the array. Intensity values from each array were scaled to a value of 1500. Scaling of the intensity values from each array to a common value is routinely used by Affymetrix as a normalization method prior to comparing data from different samples. Data preprocessing and statistical analyses were performed with SAS Statistical Software (SAS Institute Inc., SAS Campus Drive, Cary, NC) and S-Plus statistical software (Insightful Corp., Seattle, WA). Genes were excluded from the analyses if their expression, as defined by the Affymetrix software, was absent. Average difference values <0 were set to 0.
For hierarchical clustering, a Euclidean distance measure was used to calculate the 32 x 32 sample pairwise distance matrix. The sample gene expression profile was defined by the expression values for 7129 genes not excluded as described above. Each sample profile was standardized by subtracting, from each measurement within that profile, that sample profile’s mean expression, and dividing by that profile’s SD.
For K means clustering of the 7129 non-excluded genes, no standardization was used and each gene’s profile was defined as the expression level measured across the 32 samples. K means is also a sequential procedure. A number of clusters, K, is specified by the user and K gene profiles are selected at random to represent cluster centers. Each of the 7129 genes is compared to each cluster center and assigned to the center profile it is closest to. After each gene has been assigned to one cluster center, the cluster center is replaced by the mean profile of all the genes assigned to that cluster. The process repeats itself by reassigning each of the 7129 genes to the closest, new cluster center. The center is recalculated and genes reassigned repeatedly until no genes are reassigned after new cluster centers have been calculated.
For individual transcripts, their fold change in expression was the ratio of the mean expression levels of involved versus normal or uninvolved versus normal skin samples as provided by dchip (http://www.dchip.org) (92).
Molecular genotyping of HLA-C
HLA-C locus typing was performed with the C-locus specific primers 5CIn1-61 (5'-AGC GAG GG/TG CCC GCC CGG CGA-3') and 3BCIn3-12 (5'-GGA GAT GGG GAA GGC TCC CCA CT-3') described elsewhere (63). The PCR products of HLA-C span from introns 1 to 3. The PCR products were dotted onto nylon membranes and hybridized with sets of sequence-specific oligonucleotide probe (SSOP) matching sequences of HLA-A (67 SSOPs), -B (99 SSOPs) and -C (57 SSOPs) as described by Cao et al. (64). Additional group-specific amplifications and hybridizations with selected SSOPs were performed to achieve resolution of ambiguous genotypes (64). Alleles were assigned on the basis of their hybridization patterns with these SSOPs.
|
ACKNOWLEDGEMENTS |
|---|
We acknowledge the help of Dr Mark Watson and the Washington University Siteman Cancer Center Multiplexed Gene Analysis Core. We also thank Mrs Mary Akin for help with preparation of the manuscript and Dr Michael Lovett for valuable comments. We are also indebted to the physicians, their staff and psoriatic patients plus normal controls for skin biopsy donation.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed at: Washington University, 4566 Scott Avenue, Box 8232, St Louis, MO 63110, USA. Tel: +1 314 747 3264; Fax: +1 314 747 2489; Email: bowcock@genetics.wustl.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Bhalerao, J. and Bowcock, A.M. (1998) The genetics of psoriasis: a complex disorder of the skin and immune system. Hum. Mol. Genet., 7, 1537–1545.
2 Menter, A. and Barker, J. (1991) Psoriasis in practice. Lancet, 338, 231–234.[ISI][Medline]
3 Espinoza, L.R., Vansolingen, R., Cuellar, M.L. and Angulo, J. (1998) Insights into the pathogenesis of psoriasis and psoriatic arthritis. Am. J. Med. Sci., 316, 271–276.[ISI][Medline]
4 Baker, B.S., Bokth, S., Powles, A., Garioch, J.J., Lewis, H., Valdimarsson, H. and Fry, L. (1993) Group A streptococcal antigen-specific T lymphocytes in guttate psoriatic lesions. Br. J. Dermatol., 128, 493–499.[ISI][Medline]
5 Duvic, M. (1990) Immunology of AIDS related to psoriasis. J. Invest. Dermatol., 95, 38S–40S.[Medline]
6 Breuer-McHam, J.N., Marshall, G.D., Lewis, D.E. and Duvic, M. (1998) Distinct serum cytokines in AIDS-related skin diseases. Viral Immunol., 11, 215–220.[ISI][Medline]
7 Russell, T., Schultes, L. and Kuban, D. (1972) Histocompatibility (HLA) antigens associated with psoriasis. N. Engl. J. Med., 287, 738–740.
8 White, S., Newcomer, V., Mickey, M. and Terasaki, P. (1972) Disturbance of HLA antigen frequency in psoriasis. N. Engl. J. Med., 287, 740–743.
9 Fernandez-Vina, M.A., Tomfohrde, J., Silverman, A., Barnes, R., Sang, S., Young, M., Lory, D., Morris, L., Wuepper, K.D., Menter, A. and Bowcock, A. (1995) HLA genes in familial psoriasis vulgaris (pv): Evidence for genetic heterogeneity. Hum. Immunol., 40 (suppl.), C-5.2.
10 Gottlieb, A.B. and Krueger, J.G. (1990) HLA region genes and immune activation in the pathogenesis of psoriasis. Arch. Dermatol., 126, 1083–1086.[ISI][Medline]
11 Jenisch, S., Henseler, T., Nair, R.P., Guo, S.W., Westphal, E., Stuart, P., Kronke, M., Voorhees, J.J., Christophers, E. and Elder, J.T. (1998) Linkage analysis of human leukocyte antigen (HLA) markers in familial psoriasis: strong disequilibrium effects provide evidence for a major determinant in the HLA-B/-C region. Am. J. Hum. Genet., 63, 191–199.[ISI][Medline]
12 Trembath, R.C., Clough, R.L., Rosbotham, J.L., Jones, A.B., Camp, R.D., Frodsham, A., Browne, J., Barber, R., Terwilliger, J., Lathrop, G.M. and Barker, J.N. (1997) Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis. Hum. Mol. Genet., 6, 813–820.
13 Capon, F., Novelli, G., Semprini, S., Clementi, M., Nudo, M., Vultaggio, P., Mazzanti, C., Gobello, T., Botta, A., Fabrizi, G. and Dallapiccola, B. (1999) Searching for psoriasis susceptibility genes in Italy: genome scan and evidence for a new locus on chromosome 1. J. Invest. Dermatol., 112, 32–35.[ISI][Medline]
14 Tomfohrde, J., Silverman, A., Barnes, R., Fernandez-Vina, M.A., Young, M., Lory, D., Morris, L., Wuepper, K.D., Stastny, P., Menter, A. et al. (1994) Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q. Science, 264, 1141–1145.
15 Matthews, D., Fry, L., Powles, A., Weber, J., McCarthy, M., Fisher, E., Davies, K. and Williamson, R. (1996) Evidence that a locus for familial psoriasis maps to chromosome 4q. Nat. Genet., 14, 231–233.[ISI][Medline]
16 Enlund, F., Samuelsson, L., Enerback, C., Inerot, A., Wahlstrom, J., Yhr, M., Torinsson, A., Riley, J., Swanbeck, G. and Martinsson, T. (1999) Psoriasis susceptibility locus in chromosome region 3q21 identified in patients from southwest Sweden. Eur. J. Hum. Genet., 7, 783–790.[ISI][Medline]
17 Lee, Y.A., Ruschendorf, F., Windemuth, C., Schmitt-Egenolf, M., Stadelmann, A., Nurnberg, G., Stander, M., Wienker, T.F., Reis, A. and Traupe, H. (2000) Genomewide scan in german families reveals evidence for a novel psoriasis-susceptibility locus on chromosome 19p13. Am. J. Hum. Genet., 67, 1020–1024.[ISI][Medline]
18 Capon, F., Semprini, S., Dallapiccola, B. and Novelli, G. (1999) Evidence for interaction between psoriasis-susceptibility loci on chromosomes 6p21 and 1q21. Am. J. Hum. Genet., 65, 1798–1800.[ISI][Medline]
19 Nair, R.P., Henseler, T., Jenisch, S., Stuart, P., Bichakjian, C.K., Lenk, W., Westphal, E., Guo, S.W., Christophers, E., Voorhees, J.J. and Elder, J.T. (1997) Evidence for two psoriasis susceptibility loci (HLA and 17q) and two novel candidate regions (16q and 20p) by genome-wide scan. Hum. Mol. Genet., 6, 1349–1356.
20 Veal, C.D., Clough, R.L., Barber, R.C., Mason, S., Tillman, D., Ferry, B., Jones, A.B., Ameen, M., Balendran, N., Powis, S.H. et al. (2001) Identification of a novel psoriasis susceptibility locus at 1p and evidence of epistasis between PSORS1 and candidate loci. J. Med. Genet., 38, 7–13.
21 Cookson, W.O.C.M., Ubhi, B., Lawrence, R., Abecasis, G.R., Walley, A.J., Cox, H.E., Coleman, R., Leaves, N.I., Trembath, R.C., Moffatt, M.F. and Harper, J.I. (2001) Genetic linkage of childhood atopic dermatitis to psoriasis susceptibility loci. Nat. Genet., 27, 372–373.[ISI][Medline]
22 Cornelis, F., Faure, S., Martinez, M., Prud'homme, J.F., Fritz, P., Dib, C., Alves, H., Barrera, P., de Vries, N., Balsa, A. et al. (1998) New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study. Proc. Natl Acad. Sci. USA, 95, 10746–10750.
23 Lee, Y.A., Wahn, U., Kehrt, R., Tarani, L., Businco, L., Gustafsson, D., Andersson, F., Oranje, A.P., Wolkertstorfer, A., Berg, A.V. et al. (2000) A major susceptibility locus for atopic dermatitis maps to chromosome 3q21. Nat. Genet., 26, 470–473.[ISI][Medline]
24 Field, L.L., Tobias, R., Thomson, G. and Plon, S. (1996) Susceptibility to insulin-dependent diabetes mellitus maps to a locus (IDDM11) on human chromosome 14q24.3–q31. Genomics, 33, 1–8.[ISI][Medline]
25 Tomer, Y., Barbesino, G., Keddache, M., Greenberg, D.A. and Davies, T.F. (1997) Mapping of a major susceptibility locus for Graves’ disease (GD-1) to chromosome 14q31. J. Clin. Endocrinol. Metab., 82, 1645–1648.
26 Hugot, J.-P., Laurent-Puig, P., Gower-Rousseau, C., Olson, J.M., Lee, J.C., Beaugerie, L., Naom, I., Dupas, J.L., Van Gossum, A., Orholm, M. et al. (1996) Mapping of a susceptibility locus for Crohn’s disease on chromosome 16. Nature, 379, 821–823.[Medline]
27 Jawaheer, D., Seldin, M.F., Amos, C.I., Chen, W.V., Shigeta, R., Monteiro, J., Kern, M., Criswell, L.A., Albani, S., Nelson, J.L. et al. (2001) A genomewide screen in multiplex rheumatoid arthritis families suggests genetic overlap with other autoimmune diseases. Am. J. Hum. Genet., 68, 927–936.[ISI][Medline]
28 Becker, K.G., Simon, R.M., Bailey-Wilson, J.E., Freidlin, B., Biddison, W.E., McFarland, H.F. and Trent, J.M. (1998) Clustering of non-major histocompatibility complex susceptibility candidate loci in human autoimmune diseases. Proc. Natl Acad. Sci. USA, 95, 9979–9984.
29 Bowcock, A.M. and Lovett, M. (2001) Zeroing in on tolerance. Nat. Med., 7, 279–281.[ISI][Medline]
30 Robert, C. and Kupper, T.S. (1999) Inflammatory skin diseases, T cells, and immune surveillance. N. Engl. J. Med., 341, 1817–1828.
31 Travers, J.B., Hamid, Q.A., Norris, D.A., Kuhn, C., Giorno, R.C., Schlievert, P.M., Farmer, E.R. and Leung, D.Y. (1999) Epidermal HLA-DR and the enhancement of cutaneous reactivity to superantigenic toxins in psoriasis. J. Clin. Invest., 104, 1181–1189.[ISI][Medline]
32 Capuano, M., Lesnoni la Parola, I., Masini, C., Uccini, S. and Cerimele, D. (1999) Immunohistochemical study of the early histopathologic changes occurring in trauma-injured skin of psoriatic patients. Eur. J. Dermatol., 9, 102–106.[ISI][Medline]
33 Prens, E., Hooft-Benne, K., Tank, B., Van Damme, J., van Joost, T. and Benner, R. (1996) Adhesion molecules and IL-1 costimulate T lymphocytes in the autologous MECLR in psoriasis. Arch. Dermatol. Res., 288, 68–73.
34 Nickoloff, B.J., Mitra, R.S., Elder, J.T., Fisher, G.J. and Voorhees, J.J. (1989) Decreased growth inhibition by recombinant gamma interferon is associated with increased production of transforming growth factor alpha in keratinocytes cultured from psoriatic lesions. Br. J. Dermatol., 121, 161–174.[ISI][Medline]
35 Higashiyama, M., Hashimoto, K., Matsumoto, K. and Yoshikawa, K. (1994) Differential expression of transforming growth factor-alpha (TGF-alpha) and EGF receptor in transitional area of psoriatic epidermis. J. Dermatol. Sci., 7, 45–53.[Medline]
36 Van de Kerkhof, P.C., Gerritsen, M.J. and de Jong, E.M. (1996) Transition from symptomless to lesional psoriatic skin. Clin. Exp. Dermatol., 21, 325–329.[ISI][Medline]
37 Kaufman, L. (1990) Finding Groups in Data: An Introduction to Cluster Analysis, John Wiley & Sons, New York.
38 Eisen, M.B., Spellman, P.T., Brown, P.O. and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA, 95, 14863–14868.
39 Ferenczi, K., Burack, L., Pope, M., Krueger, J.G. and Austin, L.M. (2000) CD69, HLA-DR and the IL-2R identify persistently activated T cells in psoriasis vulgaris lesional skin: blood and skin comparisons by flow cytometry. J. Autoimmun., 14, 63–78.[ISI][Medline]
40 Nickoloff, B.J. (1999) The immunologic and genetic basis of psoriasis. Arch. Dermatol., 135, 1104–1110.
41 Austin, L.M., Coven, T.R., Bhardwaj, N., Steinman, R. and Krueger, J.G. (1998) Intraepidermal lymphocytes in psoriatic lesions are activated GMP-17(TIA-1)+CD8+CD3+ CTLs as determined by phenotypic analysis. J. Cutan. Pathol., 25, 79–88.[ISI][Medline]
42 Asadullah, K., Docke, W.D., Volk, H.D. and Sterry, W. (1999) The pathophysiological role of cytokines in psoriasis. Drugs of Today, 35, 913–924.[Medline]
43 Schulz, B.S., Michel, G., Wagner, S., Suss, R., Beetz, A., Peter, R.U., Kemeny, L. and Ruzicka, T. (1993) Increased expression of epidermal IL-8 receptor in psoriasis. Down-regulation by FK-506 in vitro. J. Immunol., 151, 4399–4406.[Abstract]
44 Lipshutz, R.L., Fodor, S.P.A., Gingeras, T.R. and Lockhart, D.J. (1999) High density synthetic oligonucleotide arrays. Nat. Genet., 21, 20–24.[ISI][Medline]
45 Pfundt, R., Wingens, M., Bergers, M., Zweers, M., Frenken, M. and Schalkwijk, J. (2000) TNF-alpha and serum induce SKALP/elafin gene expression in human keratinocytes by a p38 MAP kinase-dependent pathway. Arch Dermatol. Res., 292, 180–187.[ISI][Medline]
46 Jansen, B.J.H., van Ruissen, F., de Jongh, G., Zeeuwen, P.L. and Schalkwijk, J. (2001) Serial analysis of gene expression in differentiated cultures of human epidermal keratinocytes. J. Invest. Dermatol., 116, 12–22.[ISI][Medline]
47 Rasmussen, U.B, Wolf, C., Mattei, M.G., Chenard, M.P., Bellocq, J.P., Chambon, P., Rio, M.C. and Basset, P. (1993) Identification of a new interferon-alpha-inducible gene (p27) on human chromosome 14q32 and its expression in breast carcinoma. Cancer Res., 53, 4096–4101.
48 Ahn, J.Y., Tanahashi, N., Akiyama, K., Hisamatsu, H., Noda, C., Tanaka, K., Chung, C.H., Shibmara, N., Willy, P.J., Mott, J.D. et al. (1995) Primary structures of two homologous subunits of PA28, a -interferon-inducible protein activator of the 20S proteasome. FEBS Lett., 366, 37–42.[ISI][Medline]
49 Akiyama, K., Yokota, K., Kagawa, S., Shimbara, N., Tamura, T., Akioka, H., Nothwang, H.G., Noda, C., Tanaka, K. and Ichihara, A. (1994) cDNA cloning and interferon down-regulation of proteosomal subunits X and Y. Science, 265, 1231–1234.
50 Foss, G.S., Larsen, F., Solheim, J. and Prydz, H. (1998) Constitutive and interferon--induced expression of the human proteasome subunit multicatalytic endopeptidase complex-like 1. Biochim. Biophys. Acta, 1402, 17–28.[Medline]
51 Chadwick, V.S., Schlup, M.M., Ferry, D.M., Chang, A.R. and Butt, T.J. (1990) Measurements of unsaturated vitamin B12-binding capacity and myeloperoxidase as indices of severity of acute inflammation in serial colonoscopy biopsy specimens from patients with inflammatory bowel disease. Scand. J. Gastroenterol., 25, 1196–1204.[ISI][Medline]
52 Sattar, M.A. and Das, K.C. (1991) Plasma vitamin B12 binding proteins correlate with disease activity in patients with rheumatoid arthritis. Med. Lab. Sci., 48, 36–42.[ISI][Medline]
53 Vreugdenhil, G., Lindemans, J., van Eijk, H.G. and Swaak, A.J. (1992) Elevated serum transcobalamin levels in anaemia of rheumatoid arthritis: correlation with disease activity but not with serum tumour necrosis factor and interleukin 6. J. Int. Med., 231, 547–550.[ISI][Medline]
54 Iseki, T. (1993) Vitamin B12 and transcobalamin in chronic myeloproliferative disorders. Rinsho Byori, 41, 1310–1321.[Medline]
55 Marenholz, I., Zirra, M., Fischer, D.F., Backendorf, C., Ziegler, A., Mischke, D. et al. (2001) Identification of human epidermal differentiation complex (EDC)-encoded genes by subtractive hybridization of entire YACs to a gridded keratinocyte cDNA library. Genome Res., 11, 341–355.
56 South, A.P., Cabral, A., Ives, J.H., James, C.H., Mirza, G., Marenholz, I., Mischke, D., Backendorf, C., Ragoussis, J. and Nizetic D. (1999) Human epidermal differentiation complex in a single 2.5 Mbp long continuum of overlapping DNA cloned in bacteria integrating physical and transcript maps. J. Invest. Dermatol., 112, 910–918.[ISI][Medline]
57 Capon, F., Semprini, S., Chimenti, S., Fabrizi, G., Zambruno, G., Murgia, S., Carcassi, C., Fazio, M., Mingarelli, R., Dallapiccola, B. and Novelli, G. (2001) Fine mapping of the PSORS4 psoriasis susceptibility region on chromosome 1q21. J. Invest. Dermatol., 116, 728–730.[ISI][Medline]
58 Li, Q., Harju, S. and Peterson, K.R. (1999) Locus control regions coming of age at a decade plus. Trends Genet., 15, 403–408.[ISI][Medline]
59 Lawrance, I.C., Fiocchi, C. and Chakravarti, S. (2001) Ulcerative colitis and Crohn’s disease: distinctive gene expression profiles and novel susceptibility candidate genes. Hum. Mol. Genet., 10, 445–456.
60 Mahadevappa, M. and Warrington, J.A. (1999) A high-density probe array sample preparation method using 10- to 100-fold fewer cells. Nat. Biotechnol., 17, 1134–1136.
61 Fodor, S.P.A., Leighton Read, J., Pirrung, M.C., Stryer, L., Tsai Lu, A. and Solas, D. (1991) Light-directed spatially addressable parallel chemical synthesis. Science, 251, 767–773.
62 Warrington, J.A., Nair, A., Mahadevappa, M. and Tsyganskaya, M. (2000) Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol. Genomics, 2, 143–147.
63 Cereb, N. Maye, P., Lee, S., Kong, Y. and Yang, S.Y. (1995) Locus-specific amplification of HLA class I genes from genomic DNA: locus specific sequences in the first and third introns of HLA-A, -B and -C. Tissue Antigens, 45, 1.[ISI][Medline]
64 Cao, K., Chopek, M. and Fernandez-Vina, M. (1999) High and intermediate resolution DNA typing systems for class I HLA-A, -B, -C genes by hybridization with sequence-specific oligonucleotide probes (SSOP). Rev. Immunogenet., 1, 177–208.[Medline]
65 Hardas, B.D., Zhao, X., Zhang, J., Longqing, X., Stoll, S. and Elder, J.T. (1996) Assignment of psoriasin to human chromosomal band 1q21: Co-ordinate overexpression of clustered genes in psoriasis. J. Invest. Dermatol., 106, 753–758.[ISI][Medline]
66 Madsen, P., Rasmussen, H.H., Leffers, H., Honore, B., Dejgaard, K., Olsen, E., Kiil, J., Walbum, E., Andersen, A.H., Basse, B. et al. (1991) Molecular cloning, occurrence, and expression of a novel partially secreted protein "psoriasin" that is highly up-regulated in psoriatic skin. J. Invest. Dermatol., 97, 701–712.[ISI][Medline]
67 Madsen, P., Rasmussen, H.H., Leffers, H., Honore, B. and Celis, J.E. (1992) Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins. J. Invest. Dermatol., 99, 299–305.[ISI][Medline]
68 Fujimoto, W., Marvin, K.W., George, M.D., Celli, G., Darwiche, N., De Luca, L.M. and Jetten, A.M. (1993) Expression of cornifin in squamous differentiating epithelial tissues, including psoriatic and retinoic acid-treated skin. J. Invest. Dermatol., 101, 268–274.[ISI][Medline]
69 Van Ruissen, F., de Jongh, G.J., Zeeuwen, P.L., Van Erp, P.E., Madsen, P. and Schalkwijk, J.(1996) Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures. J. Cell Physiol., 168, 442–452.[ISI][Medline]
70 Schroder, J.M. and Harder, J. (1999) Human beta-defensin-2. Int. J. Biochem. Cell Biol., 31, 645–651.[ISI][Medline]
71 Mommers, J.M., van Rossum, M.M., van Erp, P.E. and van De Kerkhof, P.C. (2000) Changes in keratin 6 and keratin 10 (co-)expression in lesional and symptomless skin of spreading psoriasis. Dermatology, 201, 15–20.[ISI][Medline]
72 Komine, M., Freedberg, I.M. and Blumenberg, M. (1996) Regulation of epidermal expression of keratin K17 in inflammatory skin diseases. J. Invest. Dermatol., 107, 569–575.[ISI][Medline]
73 Troyanovsky, S.M., Leube, R.E. and Franke, W.W. (1992) Characterization of the human gene encoding cytokeratin 17 and its expression pattern. Eur. J. Cell Biol., 59, 127–137.[ISI][Medline]
74 Bonnekoh, B., Huerkamp, C., Wevers, A., Geisel, J., Sebok, B., Bange, F.C., Greenhalgh, D.A., Bottger, E.C., Krieg, T. and Mahrle, G. (1995) Up-regulation of keratin 17 expression in human HaCaT keratinocytes by interferon-gamma. J. Invest. Dermatol., 104, 58–61.[ISI][Medline]
75 Leigh, I.M., Navsaria, H., Purkis, P.E., McKay, I.A., Bowden, P.E. and Riddle, P.N. (1995) Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro. Br. J. Dermatol., 133, 501–511.[ISI][Medline]
76 Rivas, M.V., Jarvis, E.D., Morisaki, S., Carbonaro, H., Gottlieb, A.B. and Krueger, J.G. (1997) Identification of aberrantly regulated genes in diseased skin using the cDNA differential display technique. J. Invest. Dermatol., 108, 188–194.[ISI][Medline]
77 Bernard, B.A., Reano, A., Darmon, Y.M. and Thivolet, J. (1986) Precocious appearance of involucrin and epidermal transglutaminase during differentiation of psoriatic skin. Br. J. Dermatol., 114, 279–283.[ISI][Medline]
78 Takahashi, H., Kinouchi, M., Tamura, T. and Iizuka, H. (1996) Decreased beta 2-adrenergic receptor-mRNA and loricrin-mRNA, and increased involucrin-mRNA transcripts in psoriatic epidermis: analysis by reverse transcription-polymerase chain reaction. Br. J. Dermatol., 134, 1065–1069.[ISI][Medline]
79 Elder, J.T., Klein, S.B., Tavakkol, A., Fisher, G.J., Nickoloff, B.J. and Voorhees, J.J. (1990) Growth factor and proto-oncogene expression in psoriasis. J. Invest. Dermatol., 95, 7S–9S.[Medline]
80 Hussain, H., Shornick, L.P., Shannon, V.R., Wilson, J.D., Funk, C.D., Pentland, A.P. and Holtzman, M.J. (1994) Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis. Am. J. Physiol., 266, C243–C253.
81 Gan, L., Lee, I., Smith, R., Argonza-Barrett, R., Lei, H., McCuaig, J., Moss, P., Paeper, B. and Wang, K. (2000) Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region. Gene, 257, 119–130.[ISI][Medline]
82 Nickoloff, B.J., Mitra, R.S., Varani, J., Dixit, V.M. and Polverini, P.J. (1994) Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis. Am. J. Pathol., 144, 820–828.[Abstract]
83 Wraight, C.J., Edmondson, S.R., Fortune, D.W., Varigos, G. and Werther, G.A. (1997) Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in the psoriatic lesion. J. Invest. Dermatol., 108, 452–456.[ISI][Medline]
84 Wei, L., Debets, R., Hegmans, J.J., Benner, R. and Prens, E.P. (1999) IL-1 beta and IFN-gamma induce the regenerative epidermal phenotype of psoriasis in the transwell skin organ culture system. IFN-gamma up-regulates the expression of keratin 17 and keratinocyte trans glutaminase via endogenous IL-1 production. J. Pathol., 187, 358–364.[ISI][Medline]
85 Prens, E., Hegmans, J., Lien, R.C., Debets, R., Troost, R., van Joost, T. and Benner, R. (1996) Increased expression of interleukin-4 receptors on psoriatic epidermal cells. Am. J. Pathol., 148, 1493–1502.[Abstract]
86 Nakamura, K., Yasaka, N., Asahina, A., Kato, M., Miyazono, K., Furue, M. and Tamaki, K. (1998) Increased numbers of CD68 antigen positive dendritic epidermal cells and upregulation of CLA (cutaneous lymphocyte-associated antigen) expression on these cells in various skin diseases, J. Dermatol. Sci., 18, 170–180.[ISI][Medline]
87 Deleuran, M., Buhl, L., Ellingsen, T., Harada, A., Larsen, C.G., Matsushima, K. and Deleuran, B. (1996) Localization of monocyte chemotactic and activating factor (MCAF/MCP-1) in psoriasis. J. Dermatol. Sci., 13, 228–236.[ISI][Medline]
88 Figueroa, C.D., MacIver, A.G. and Bhoola, K.D. (1989) Identificatioin of a tissue kallikrein in human polymorphonuclear leucocytes. Br. J. Haematol., 72, 321–328.[ISI][Medline]
89 Creamer, D., Jaggar, R., Allen, M., Bicknell, R. and Barker, J. (1997) Overexpression of the angiogenic factor platelet-derived endothelial cell growth factor thymidine phosphorylase in psoriatic epidermis. Br. J. Dermatol., 137, 851–855.[ISI][Medline]
90 Waseem, A., Dogan, B., Tidman, N., Alam, Y., Purkis, P., Jackson, S., Lalli, A., Machesney, M. and Leigh, I.M. (1999) Keratin 15 expression in stratified epithelia: downregulation in activated keratinocytes. J. Invest. Dermatol., 112, 362–369.[ISI][Medline]
91 Basset-Seguin, N., Escot, C., Moles, J.P., Blanchard, J.M., Kerai, C. and Guilhou, J.J. (1991) C-fos and c-jun proto-oncogene expression is decreased in psoriasis: an in situ quantitative analysis. J. Invest. Dermatol., 97, 672–678.[ISI][Medline]
92 Li, C. and Wong, W.H. (2001) Model-based analysis of oligonucleotide ar