Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5

Figures & data

Table 1. Clinical and laboratory data of the patients with RA and OA who were studied for gene expression profiling in synovial tissue.

Case	Diagnosis	Age (years)	Sex	Clinical activity			Histological activity		Molecular activity
				RF	CRP (mg/L)		Krenn
S17	RA	63	F	+	14	H	8	H	H
S20	RA	64	M	+	36	H	8	H	H
S49	RA	54	F	+	30	H	7	H	H
S52	RA	85	F	ND	18	H	7	H	H
S56	RA	62	F	+	42	H	6	H/L	H
S57	RA	79	F	+	24	H	7	H	H
S68	RA	73	F	+	127	H	7	H	H
S69	RA	52	F	+	22	H	9	H	H
S11	RA	73	M	+	0	L	5	L	L
S53	RA	78	F	ND	0	L	4	L	L
S65	RA	71	F	ND	0	L	5	L	L
S13	OA	68	F	–	0		3
S40	OA	79	F	–	0		2
S62	OA	76	M	–	0		2
S63	OA	63	F	–	0		2
S66	OA	75	F	–	2		3

RF, Rheumatoid factor; CRP, C-reactive protein; Krenn, synovitis score; H, high inflammation RA; L, low inflammation RA; ND, not determined.

Figure 1. Microdissection of the synovial lining in RA. (A) After toluidine blue staining, a section was subjected to LMD. The sample was observed on the PC monitor and the lining area was marked. (B) The sample was cut with a laser beam along the defined line. (C) After microdissection, the sample was collected by gravity into a tube cap. In this manner, we obtained more than 5000 cells from the RA or OA synovial lining sections.

Figure 2. Gene-expression clustering in RA and OA. (A) Cluster with 197 genes (after SAM analysis of normalized 14 519 genes; criterion: FC RA/OA ≥ 2.0 or ≤ 0.5, FDR < 0.001). The dendrogram shows the major groups, that is RA (subgroups: high RA and low RA) and OA groups. (B) Heatmap of selected genes. Expression of chemokines (CCL5 and CXCL9/10) and transcription factors (IRF1 and STAT1) were selected and analysed using GeneSpring software. The levels of these genes are more strongly up-regulated in RA than in OA. The colour intensity indicates the magnitude of the deviation from the median (yellow). Red represents relative expression levels higher than the median level across all tissues and blue represents lower expression levels.

Figure 3. List of top 20 up- and down-regulated genes from the filtered 197 genes. The upper panel shows the top 20 genes from 121 up-regulated genes (RA/OA) on the basis of Fold Change (FC). The lower panel is a list of the bottom 20 genes from 76 down-regulated genes.

Figure 4. Gene network generated through the use of Ingenuity Pathway Analysis (IPA). IPA was applied to TNF, chemokines (CCL5, CXCL9/10), and transcription factor (STAT1 and IRF1) genes to create a network inter-related to inflammatory mediator genes. Interacting nodes are defined by either direct relationships, which require direct physical contact (solid arrows), or indirect relationships (dashed arrows). The direction of the arrows shows the direction of the interaction. The blue line indicates the connection with STAT1. Red molecules indicate higher expression in RA than in OA at the gene level.

Table 2. List of up- and down-regulated genes (chemokines and transcription factors).

Probe ID	Gene symbol	Accession no.	Gene name	RA vs. OA
				Fold Change	p-value
Chemokine
5570278	CXCL9	NM_002416.1	Chemokine (C-X-C motif) ligand 9	5.92	0.0001
7570408	CCL5	NM_002985.2	Chemokine (C-C motif) ligand 5	4.37	0.0002
620717	CCL5	NM_002985.2	Chemokine (C-C motif) ligand 5	3.39	0.0005
6270553	CXCL10	NM_001565.2	Chemokine (C-X-C motif) ligand 10	3.39	0.0016
4250053	CCL3L1	NM_021006.4	Chemokine (C-C motif) ligand 3-like 1	0.35	0.0014
4610615	CXCL12	NM_000609.4	Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)	0.32	0.0004
6590682	CCL3	NM_002983.1	Chemokine (C-C motif) ligand 3	0.30	0.0013
Transcription factor
2570079	STAT1	NM_139266.1	Signal transducer and activator of transcription 1.91 kDa (β-type)	3.79	0.0001
1820750	STAT1	NM_007315.2	Signal transducer and activator of transcription 1.91 kDa (common)	2.56	0.0001
4810187	STAT1	NM_007315.2	Signal transducer and activator of transcription 1.91 kDa (α-type)	2.09	0.0001
6250064	IRF1	NM_002198.1	Interferon regulatory factor 1	2.13	0.0001

Figure 5. Immunohistochemical staining of the synovium from (A, C, E, F, and G) RA and (B and D) OA patients. (A and B) anti-CCL5; (C and D) anti-STAT1; (E) anti-IRF1; (F) anti-CXCL9; (G) anti-CXCL10. Significant presence of all antigens was detected in the synovial lining cells of RA but not of OA.

Figure 6. Relationship between STAT1 and CCL5 established by immunohistochemical analysis. Expression of STAT1 and CCL5 at the protein level is higher in the synovial cells of RA (n = 25) than in those of OA (n = 10). The STAT1 score correlates significantly with the CCL5 score (r = 0.93, p < 10^–6).