NO is a freely diffusible radical gas, which is a product of the catalytic conversion of L-arginine to L-citrulline by nitric oxide synthases (NOS) (EC 1.14.13.39) via the chemical reaction between the guanidino-nitrogen of L-arginine and dioxygen. The activity of the inducible NO synthase (iNOS) requires pro-inflammatory cytokines such as IL-1 and TNF-α for upregulation of mRNA and protein. The activity of iNOS, in turn, is under strict control of nicotinamide adenine dinucleotide phosphate (NADPH), flavine adenidine dinucleotide, flavin mononucleotide, heme and 5,6,7,8-tetrahydrobiopterin for activity [28]. The iNOS is highly expressed in the rheumatoid synovium, particularly in synovial fibroblasts [29,30]. The mRNA initiation site of the iNOS gene is preceded by a promoter sequence box, along with two distinct regions upstream containing consensus sequences for the binding of various transcription factors. Region 1 contains lipopolysaccharide responsive elements such as the binding sites for nuclear factor-1, IL-6 and NF-κ B, indicating a locus for LPS induced synthesis of iNOS. Region 2 contains motifs for interferon gamma (IFN-γ)-regulated transcription factors but does not directly regulate induction of iNOS; instead, it subserves region 1. LPS therefore stimulates iNOS synthesis directly, and IFN-γ acts in synergy with LPS to augment iNOS synthesis and NO production. This synergy also extends to the cytokines IL-1 and TNF-α, which, in combination with IFN-γ, augment the synthesis of iNOS and NO [31]. Apoptosis induced by NO is associated with nuclear p53 protein expression in cultured human fibroblasts [32]. NO can also induce the synthesis and activity of cyclo-oxygenase (COX)-2 and hemeoxygenase (HO)-1.

Prostaglandin H₂ (PGH₂) synthase (EC 1.14.99.1) has two activities, COX and peroxidase, and occurs in two isoforms, known as COX-1 and COX-2 [33]. The inducible COX-2 mRNA and protein are stimulated largely by the same factors as iNOS, such as IL-1, TNF-α and IFN-γ [34]. The promoter region of COX-2 contains binding sites for NF-κ B, NF-IL6 and two motifs for IFN-γ activatedsequences [34]. PGE₂ and PGE₁ inhibit cytokine-induced metalloproteinase expression in human synovial fibroblasts [35]. COX inhibition conversely enhances the production of pro-MMP-1 in human rheumatoid synovial fibroblasts [36]. PGE₂ also enhances the synthesis of IL-8 and IL-6, but inhibits granulocyte-macrophage colony-stimulating factor production by IL-1 stimulated synovial fibroblasts [37].

Carbon monoxide is produced by two homologous microsomal HO (EC 1.14.93) isoenzymes: inducible HO-1 (heat shock protein-32) and constitutively expressed HO-2. The latter is widely expressed in fibroblasts, and HO-1 can be induced in these and other cell types by hypoxia and free radicals [38]. HO-1 prevents cell death by regulating intracellular iron levels. HO functions by cleaving heme to biliverdin and carbon monoxide, in the presence of NADPH and NADPH-cytochrome P₄₅₀, with equimolar iron released from the heme as a co-product [39]. There is a regulatory loop between iron metabolism and the NO pathway: intracellular ferric iron (Fe³⁺) levels can significantly decrease iNOS mRNA transcription, and iron chelating agents like desferrioxamine can increase iNOS transcription and NO production [40]. NO itself, conversely, can directly control intracellular iron metabolism by activating the iron-regulatory protein involved in ferritin translocation. Therefore, the interplay between iNOS and HO-1 activities may have far-reaching consequences in situations characterized by oxidative stress.

The ECM-cell interactions are coupled to cytoskeletal elements, such as α -actinin, talin and tensin, and affect various tyrosine kinases, for example focal adhesion kinases, Src (the protein product of the src gene of the Rous sarcoma virus) family kinases and Crk (the protein product of the crk gene from chicken retroviruses CT10 and ASV-1). Focal adhesion kinases provide a potent anoikis resistance factor [41], anoikis referring to apoptosis caused by loss of ECM-cell adhesion. ECM-fibroblast interactions are important because the synovial lining cells and the pannus are subjected to shear stress. Synovial cells are also subjected to cyclic mechanical loading during the movement of the joint.

The synovial ECM provides hydraulic resistance, preventing rapid seepage of synovial fluid out of the joint cavity, and modifies the traffic of macromolecules. It may trap antigens, which contribute to inflammation. The three main classes of synovial structural polymers are collagen (scaffolding), extrafibrillar glycosaminoglycans/proteoglycans and structural glycoproteins. Under normal circumstances, collagen is hidden in a matrix created by the latter two classes. Fibronectin guides fibroblast migration as an immobilized substrate and attractant in the leading edge of the pannus (haptotaxis) [42]. The extra domain-A fibronectin isoform is associated with the activated, transformed state of type B lining cells [43]. The interaction between connecting sequence-1 fibronectin (or vascular cell adhesion molecule-1) and α 4β 1 (very late activation antigen-4) may play a role in the proliferation of synovial lining and lymphocyte migration [44]. EMMPRIN (M_r = 58 000) is an integral plasma membrane glycoprotein of the pericellular matrix belonging to the immunoglobulin superfamily, previously referred to as tumor-cell derived collagenase stimulatory factor. It is identical to the M6 leukocyte activation antigen, and highly homologous to rat OX-47 or CE9, mouse basigin or gp42, and chicken HT-7 or neurothelin molecules. Reciprocal immunoprecipitation, cell surface crosslinking and immunofluorescence co-localization experiments demonstrated that EMMPRIN can form a complex with integrins α 3β 1 and α 6β 1, which may play a role in the synovial membrane [45]. Many other ECM-fibroblast interactions are of potential relevance in the synovial membrane (Table 3) [46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61].

It has been claimed that the rheumatoid arthritis synovial fibroblasts differ from their nonrheumatoid counterparts in terms of growth rate, life span, glycolytic metabolism, synthesis of hyaluronan and sulfated glycosaminoglycans, acid hydrolase activities, and metabolic and structural mitochondrial proteins [1]. The rheumatoid fibroblasts show a sustained and distinct morphology and pattern of gene activation [74,75], and might represent nonrheumatoid fibroblasts, but might also be phenotypically altered chondrocytes or bone marrow derived stem cells. These differences between the normal and the inflammatory synovium may be due to a selection pressure in the synovial mileau, where water- and lipid-soluble stimuli, cyclic loading, shear stress, ECM contacts and direct cell-cell contacts more or less permanently modulate the phenotype and function of fibroblast-like cells in the synovial lining, stroma and pannus.