title: Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion.
abstract: BACKGROUND
  Increased numbers of mast cells (MCs) are found in the synovial tissues and fluids of patients with rheumatoid arthritis (RA), and at sites of cartilage erosion. MC activation has been reported for a significant proportion of rheumatoid specimens. Because the MC contains potent mediators, including histamine, heparin, proteinases, leukotrienes and multifunctional cytokines, its potential contributions to the processes of inflammation and matrix degradation have recently become evident. Proinflammatory cytokines are important mediators of inflammation, immunity, proteolysis, cell recruitment and proliferation. Tumour necrosis factor (TNF) reportedly plays a pivotal role in the pathogenesis o RA, especially its ability to regulate interleukin (IL)-1beta expression, this being important for the induction of prostanoid and matrix metalloproteinase production by synovial fibroblasts and chondrocytes. IL-15 has been assigned numerous biological effects and has been assigned numerous biological effects and has been implicated as an important factor in TNF-alpha expression by monocyte/macrophages. Some in vitro studies have placed IL-15 upstream from TNF-alpha in the cytokine cascade, suggesting an interdependence between TNF, IL-1 and IL-15 for the promotion of proinflammatory cytokine expression in the rheumatoid joint.
  
  
  OBJECTIVE
  To examine the in situ relationships of TNF-alpha, IL-1beta and IL-15 in relation to MC activation in rheumatoid tissues by use of immunolocalization techniques; and to compare quantitatively the proinflammatory cytokine production by specific cell cultures and rheumatoid synovial explants with and without exposure to a MC secretagogue.
  
  
  METHODS
  Samples of rheumatoid synovial tissue and cartilage-pannus junction were obtained from patients (n=15) with classic late-stage RA. Tissue sections were immunostained for MC (tryptase) and the proinflammatory cytokines IL-1, TNF-alpha and IL-15. Rheumatoid synovial tissue explants were cultured in Dulbecco's modified Eagles medium (DMEM) containing either the MC secretagogue rabbit antihuman immunoglobulin (Ig)E, or control rabbit IgG. Primary rheumatoid synovial cell cultures, human articular chondrocytes, synovial fibroblasts and synovial macrophages were prepared as described in the full article. Conditioned culture media from these cultures were collected and assayed for IL-1beta, TNF-alpha and IL-15 using enzyme-linked immunosorbent assay methodology.
  
  
  RESULTS
  Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Sites of MC activation were associated with localized oedema, and TNF-alpha, IL-1alpha and IL-1beta production by a proportion of mononuclear inflammatory cells. By contrast, no evidence was found for IL-15 production in tissue sites containing either intact or activated MCs, and IL-15 expression, when observed, bore no relation to tissue sites where TNF-alpha and IL-1beta were evident. The immunodetection of IL-15 was restricted to microfocal sites and was not typical of most junctional specimens, but was associated with a proportion of articular chondrocytes in a minority of junctional specimens. MC activation within synovial explant cultures was induced by the addition of polyclonal antibody to human IgE. MC activation significantly reduced the levels of TNF-alpha and IL-1beta released into the medium, this representing approximately 33% of control values. By contrast, MC activation had little effect of the levels of IL-15 released into the culture medium, the average value being very low in relation to the release of TNF-alpha and IL-1beta. Thus, induced MC activation brings about changes in the amounts of released tryptase, TNF-alpha and IL-1beta, but not of IL-15. Four preparations of primary rheumatoid synovial cell cultures produced more IL-1beta than TNF-alpha, with only modest values for IL-15 production, indicating that all three cytokines are produced and released as free ligands by these cultures. Of specific cell types that produced IL-15 in vitro, macrophages produced more than fibroblasts, which in turn produced more than chondrocytes. This demonstrates that all three cell types have the potential to produce IL-15 in situ.
  
  
  CONCLUSIONS
  The biological consequences of MC activation in vivo are extremely complex, and in all probability relate to the release of various combinations of soluble and granular factors, as well as to the expression of appropriate receptors by neighbouring cells. The subsequent synthesis and release of cytokines such as TNF-alpha and IL-1 may well follow at specific stages after activation, or may be an induced cytokine response by adjacent macrophagic or fibroblastic cells. However, because no IL-15 was detectable either in or around activated or intact MCs, and the induced MC activation explant study showed no change in IL-15 production, it seems unlikely that the expression of this cytokine is regulated by MCs. The immunohistochemistry (IHC) demonstration of IL-15 at sites of cartilage erosion, and especially by some chondrocytes of articular cartilage, showed no spatial relationship with either T cells or neutrophils, and suggests other functional properties in these locations. The lack of evidence for an in situ association of IL-15 with TNF and IL-1 does not support a role for IL-15 in a proinflammatory cytokine 'cascade', as proposed by other in vitro experiments. We believe that sufficient evidence is available, however, to suggest that MC activation makes a significant contribution to the pathophysiological processes of the rheumatoid lesion.
authors.affiliation: University Department of Medicine, Manchester Royal Infirmary, UK. david.woolley@mri.cmht.nwest.nhs.uk
authors.forename: D E
authors.initials: DE
authors.lastname: Woolley
authors.forename: L C
authors.initials: LC
authors.lastname: Tetlow
mesh_terms: D001172:Arthritis, Rheumatoid / Q000276:immunology* / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000378:metabolism*; D019409:Interleukin-15 / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D008407:Mast Cells / Q000276:immunology* / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D007375:Interleukin-1; D019409:Interleukin-15; D014409:Tumor Necrosis Factor-alpha