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Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion.

Woolley DE, Tetlow LC - Arthritis Res. (2000)

Bottom Line: MC activation significantly reduced the levels of TNF-alpha and IL-1beta released into the medium, this representing approximately 33% of control values.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Department of Medicine, Manchester Royal Infirmary, UK. david.woolley@mri.cmht.nwest.nhs.uk

ABSTRACT

Introduction: 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.

Aims: 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.

Materials and 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.

Discussion: 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.

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Immunolocalisation of mast cell tryptase, tumour necrosis factor				(TNF)-α, interleukin (IL)-1α and IL-15 in the rheumatoid lesion.				(a) Micrograph showing mast cell tryptase (red) with extracellular				staining indicative of mast cell (MC) activation associated with localized				oedema (arrow) and TNF-α expression (brown) by a proportion of mononuclear				cells. (b) Micrograph showing mast cell tryptase (red), local MC				activation and associated mononuclear cells stained for IL-1α (brown).				(c) Low power micrograph showing distribution of mast cells (tryptase,				red) and their degranulation in rheumatoid synovial tissue. (d)				Consecutive section to (c) stained for IL-15. Note the absence of IL-15				from MC activation sites. (e) Micrograph of cartilage-pannus junction showing				both extracellular and intracellular staining for IL-15 (red). Note the				microfocal nature of cytokine production by a minority of cells. (f)				Micrograph of rheumatoid lesion showing chondrocytic cells stained for				IL-15 (red) with little evidence of cytokine production by the overlying pannus				tissue (to right of micrograph). Bars: (a) and (b) 35 μm;				(c) and (d) 120 μm; (e) 35 μm; and (f)				45 μm.
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Figure 1: Immunolocalisation of mast cell tryptase, tumour necrosis factor (TNF)-α, interleukin (IL)-1α and IL-15 in the rheumatoid lesion. (a) Micrograph showing mast cell tryptase (red) with extracellular staining indicative of mast cell (MC) activation associated with localized oedema (arrow) and TNF-α expression (brown) by a proportion of mononuclear cells. (b) Micrograph showing mast cell tryptase (red), local MC activation and associated mononuclear cells stained for IL-1α (brown). (c) Low power micrograph showing distribution of mast cells (tryptase, red) and their degranulation in rheumatoid synovial tissue. (d) Consecutive section to (c) stained for IL-15. Note the absence of IL-15 from MC activation sites. (e) Micrograph of cartilage-pannus junction showing both extracellular and intracellular staining for IL-15 (red). Note the microfocal nature of cytokine production by a minority of cells. (f) Micrograph of rheumatoid lesion showing chondrocytic cells stained for IL-15 (red) with little evidence of cytokine production by the overlying pannus tissue (to right of micrograph). Bars: (a) and (b) 35 μm; (c) and (d) 120 μm; (e) 35 μm; and (f) 45 μm.

Mentions: Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Previous studies have shown evidence of MC activation in situ, as judged by the release of the MC-specific enzyme tryptase. Figure 1a shows MC activation with evidence of local oedema, associated with sites of TNF-α production by a proportion of mononuclear inflammatory cells. Figure 1b also shows dual immunolocalization of MC tryptase together with IL-1β production by some neighbouring cells. Associations of IL-1β and sites of MC activation were similarly observed [2]. Although MCs are reported to have the potential to express TNF-α and IL-1, only occasionally has TNF-α production by MCs been demonstrated in our rheumatoid specimens.


Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion.

Woolley DE, Tetlow LC - Arthritis Res. (2000)

Immunolocalisation of mast cell tryptase, tumour necrosis factor				(TNF)-α, interleukin (IL)-1α and IL-15 in the rheumatoid lesion.				(a) Micrograph showing mast cell tryptase (red) with extracellular				staining indicative of mast cell (MC) activation associated with localized				oedema (arrow) and TNF-α expression (brown) by a proportion of mononuclear				cells. (b) Micrograph showing mast cell tryptase (red), local MC				activation and associated mononuclear cells stained for IL-1α (brown).				(c) Low power micrograph showing distribution of mast cells (tryptase,				red) and their degranulation in rheumatoid synovial tissue. (d)				Consecutive section to (c) stained for IL-15. Note the absence of IL-15				from MC activation sites. (e) Micrograph of cartilage-pannus junction showing				both extracellular and intracellular staining for IL-15 (red). Note the				microfocal nature of cytokine production by a minority of cells. (f)				Micrograph of rheumatoid lesion showing chondrocytic cells stained for				IL-15 (red) with little evidence of cytokine production by the overlying pannus				tissue (to right of micrograph). Bars: (a) and (b) 35 μm;				(c) and (d) 120 μm; (e) 35 μm; and (f)				45 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC17805&req=5

Figure 1: Immunolocalisation of mast cell tryptase, tumour necrosis factor (TNF)-α, interleukin (IL)-1α and IL-15 in the rheumatoid lesion. (a) Micrograph showing mast cell tryptase (red) with extracellular staining indicative of mast cell (MC) activation associated with localized oedema (arrow) and TNF-α expression (brown) by a proportion of mononuclear cells. (b) Micrograph showing mast cell tryptase (red), local MC activation and associated mononuclear cells stained for IL-1α (brown). (c) Low power micrograph showing distribution of mast cells (tryptase, red) and their degranulation in rheumatoid synovial tissue. (d) Consecutive section to (c) stained for IL-15. Note the absence of IL-15 from MC activation sites. (e) Micrograph of cartilage-pannus junction showing both extracellular and intracellular staining for IL-15 (red). Note the microfocal nature of cytokine production by a minority of cells. (f) Micrograph of rheumatoid lesion showing chondrocytic cells stained for IL-15 (red) with little evidence of cytokine production by the overlying pannus tissue (to right of micrograph). Bars: (a) and (b) 35 μm; (c) and (d) 120 μm; (e) 35 μm; and (f) 45 μm.
Mentions: Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Previous studies have shown evidence of MC activation in situ, as judged by the release of the MC-specific enzyme tryptase. Figure 1a shows MC activation with evidence of local oedema, associated with sites of TNF-α production by a proportion of mononuclear inflammatory cells. Figure 1b also shows dual immunolocalization of MC tryptase together with IL-1β production by some neighbouring cells. Associations of IL-1β and sites of MC activation were similarly observed [2]. Although MCs are reported to have the potential to express TNF-α and IL-1, only occasionally has TNF-α production by MCs been demonstrated in our rheumatoid specimens.

Bottom Line: MC activation significantly reduced the levels of TNF-alpha and IL-1beta released into the medium, this representing approximately 33% of control values.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Department of Medicine, Manchester Royal Infirmary, UK. david.woolley@mri.cmht.nwest.nhs.uk

ABSTRACT

Introduction: 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.

Aims: 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.

Materials and 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.

Discussion: 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.

Show MeSH
Related in: MedlinePlus