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- Materials and methods
- Conflict of interest
CD4+ T cells can develop into T helper cells (Th1 and Th2 cells) characterized by the production of different cytokines, such as interleukin-2 (IL-2) and interferon-γ from Th1 cells, and IL-4 and IL-13 from Th2 cells. Th2 cytokines are associated with allergic reactions and maturation of mast cells, but have recently also been implicated in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) (Pedotti et al., 2003). Mast cells are involved in allergy as well as in innate and acquired immunity (Galli et al., 2005), including T-cell-mediated disorders (Gregory et al., 2006) and autoimmune disorders (Benoist and Mathis, 2002). Mast cells and T cells could interact in a variety of immune responses (Pedotti et al., 2003; Nakae et al., 2005), including many inflammatory diseases (Theoharides and Kalogeromitros, 2006). Mast cells are also located at the blood–brain barrier (BBB), especially in the choroid plexus, diencephalon and the third ventricle (Silver et al., 1996), as well as at MS plaques (Ibrahim et al., 1996). There, mast cells could regulate BBB permeability (Esposito et al., 2001), disruption of which precedes clinical or pathological signs of MS (Stone et al., 1995); this could attract T cells and superactivate them.
Multiple sclerosis is the second most common neurological disorder leading to severe disability in almost half a million people in the United States (Noseworthy et al., 2000). MS is characterized by inflammation and demyelination of the CNS mediated by infiltration of CD4+ Th1 cells, macrophages, B cells and mast cells (Frohman et al., 2006). However, it is still not known how T cells enter the brain and are sensitized to induce brain inflammation. This process is dependent on several factors, including IL-8, which regulates recruitment and activation of leukocytes, as well as the expression of vascular adhesion molecules that permit leukocyte transmigration (Mirowska-Guzel et al., 2006). Involvement of mast cells in the pathophysiology of MS is based on both anatomical and biochemical evidence (Krüger, 2001; Brown et al., 2002). Mast cell tryptase is elevated in the CSF of MS patients (Rozniecki et al., 1995), and can cause widespread inflammation by stimulating protease-activated receptors (Molino et al., 1997). Release of myelin basic protein (MBP) or other myelin breakdown products could also induce rat mast cell degranulation (Johnson et al., 1988; Theoharides et al., 1993). Moreover, genes highly upregulated in MS plaques include mast cell-associated molecules, such as tryptase, the IgE receptor (FcɛRI) and the histamine H1 receptor (Lock et al., 2002).
Current available MS therapies are not curative. Certain naturally occurring flavonoids (Kimata et al., 2000; Middleton et al., 2000; Kumazawa et al., 2006) inhibit the release of pro-inflammatory molecules from human mast cells (Kempuraj et al., 2005) and can suppress EAE (Hendriks et al., 2004). Here, we investigated whether human mast cells can affect Jurkat T-cell activation either through direct contact and/or mediators released in response to stimulation by MBP, and whether these processes could be blocked by pretreatment with the flavonoid, luteolin.
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- Materials and methods
- Conflict of interest
Here, we show that MBP stimulated human mast cells to release the pro-inflammatory mediators, IL-6, IL-8, TGF-β1, TNF-α, VEGF, histamine and tryptase. The amounts released were less than after allergic stimulation. MBP and myelin fragment P2 had been previously shown to induce rat mast cell degranulation (Johnson et al., 1988; Theoharides et al., 1993), and led to homogeneic brain demyelination, but at millimolar concentrations (Theoharides et al., 1993). The MBP concentration stimulating human cultured mast cells were still higher than the levels expected to be present in CSF of MS patients (Sellebjerg et al., 1998; Ohta and Ohta, 2002). However, brain mast cells may respond differently to MBP compared with the fairly undifferentiated human cultured mast cells.
We also showed that activated Jurkat cells were stimulated by normal human cultured mast cells through direct contact, as well as through TNF-α released from mast cells in response to MBP. We present the IL-2 release results in Figure 3 as fold change because there is decreased IL-2 production from those Jurkat cells, as explained in the Results section. Even though the absolute amounts of IL-2 released is lower, the addition of hCBMCs to activated Jurkat cells still releases about 25 times, compared with 30 times in the other experiments, more IL-2 than Jurkat cells alone. Activated Jurkat cells also induced mast cell release of IL-8. Luteolin inhibits MBP-induced mast cell mediator release, as well as IL-2 release from Jurkat cells, whether activated only by anti-CD3/anti-CD28 or by mast cells. Our finding of the importance of cell-to-cell contact is supported by previous results using rodent mast cells. Soluble TNF increased the surface expression of OX40, ICOS, PD-1 and other co-stimulatory molecules on CD3+ T cells (Nakae et al., 2005). Expression of mast cell co-stimulatory molecules, OX40 ligand and 4-1BB ligand, was enhanced by IgE activation and promoted T-cell activation through cell–cell contact (Gregory et al., 2006). Co-culture of leukaemic mast cells with activated but not resting T cells promoted marked adhesion of mast cells to vascular adhesion molecule-1 (Brill et al., 2004).
Luteolin effectively inhibited MBP-induced mast cell mediator release at 10 and 100 μM, whereas it exhibited a dose-dependent inhibition (1–100 μM) of mast cell-induced IL-2 release. Flavonoids comprise a group of polyphenolic compounds naturally occurring in fruits, vegetables, nuts, seeds, herbs, spices and red wine with antioxidant properties (Middleton et al., 2000). Flavonoids are potent scavengers of reactive oxygen species that also has a prominent function in MS (Lu et al., 2000) and EAE (Ruuls et al., 1995). Luteolin, a flavone analogue of quercetin, inhibits IgE-mediated release of histamine, leukotrienes, prostaglandin D2 and granulocyte-macrophage colony-stimulating factor from hCBMCs (Kimata et al., 2000). Luteolin also reduces inflammation and axonal damage in the CNS by preventing monocyte migration across the brain endothelium (Hendriks et al., 2004) and inhibits clinical symptoms of EAE through the inhibition of macrophage myelin phagocytosis (Hendriks et al., 2004). Luteolin also inhibits in vitro antigen-specific proliferation and interferon-γ production by murine and human autoimmune T cells (Verbeek et al., 2004). Regarding its mechanism of action, luteolin may inhibit mast cell activation through the inhibition of Ca2+ influx and PKC activation, as histamine and cytokines production are regulated by intracellular Ca2+ levels (Kimata et al., 2000). It was also shown that quercetin inhibited the activation of PKC-θ involved in IL-1-induced IL-6 release from hCBMCs (Kempuraj et al., 2005). Flavonoids, including luteolin, also inhibit 15-lipoxygenase-1, which may contribute to their antioxidant and anti-inflammatory activity (Sadik et al., 2003).
Mast cells secrete a wide variety of potent chemical mediators that can initiate and modulate several inflammatory pathways (Galli et al., 2005; Theoharides and Kalogeromitros, 2006), including T-cell activation (Mekori and Metcalfe, 2000). In fact, EAE, an animal model of MS, was reduced and delayed in mast cell-deficient W/Wv mice (Brown et al., 2002) and mast cells were required for optimal T-cell responses in this model (Gregory et al., 2005). Mast cells stimulated by FcɛRI aggregation release TNF-α (Nakae et al., 2006) and could activate T cells (Bongioanni et al., 2000a; Gregory et al., 2006). Mast cells represent a major potential source of TNF-α, which influences T-cell recruitment and activation (Tartaglia et al., 1993) in MS. Moreover, mast cell-derived TNF-α can promote neutrophil recruitment (Nakae et al., 2007). Mast cell-derived histamine and TNF-α increase microvascular permeability, leukocyte rolling and adhesion, thus contributing to the infiltration of T cells and monocytes into the CNS in MS.
The sites crucial in antigen entry are trafficked by CD4+ T cells. Mast cells can interact with T cells to amplify the magnitude of immune responses elicited in sensitized hosts at sites of antigen challenge. In terms of MS, the most likely place for contact, especially because mast cells do not circulate like T cells, would be at the BBB around which mast cells are critically located (Theoharides et al., 1993). In the CNS, mast cells are mainly found in the leptomeninges, choroid plexus and the median eminence (Silver et al., 1996), In fact, the mast cell had been proposed to act as ‘the immune gate to the brain’ (Theoharides, 1990), by regulating the permeability of the BBB (Esposito et al., 2001), through the activation of corticotrophin-releasing factor receptors (Esposito et al., 2002). Disruption of the BBB precedes any clinical or pathological signs of MS (Stone et al., 1995; Minagar and Alexander, 2003). Mast cells express corticotrophin-releasing factor receptors, activation of which leads to selective release of VEGF (Cao et al., 2005). This fact led to the premise that corticotrophin-releasing factor released under stress regulates BBB permeability (Theoharides and Konstantinidou, 2007). In this study, we report that MBP induces VEGF release from mast cells. VEGF was upregulated in MS plaques (Prescholdt et al., 2002) and in serum of MS patients (Su et al., 2006). In fact, serum levels of VEGF correlated with disease activity in autoimmune diseases (Carvalho et al., 2007).
We also showed that MBP induced histamine and tryptase release. CSF levels of histamine were elevated in MS patients with remitting and progressive disease (Tuomisto et al., 1983). Mast cell proteases are potent myelinolytic agents (Dietsch and Hinrichs, 1991) and can cause direct myelin damage (Johnson et al., 1988). Tryptase is elevated in CSF of MS patients (Rozniecki et al., 1995), and could activate peripheral blood mononuclear cells isolated from MS patients to synthesize and release IL-1β, IL-6 and TNF-α (Malamud et al., 2003), both involved in the pathogenesis of MS. Tryptase could also activate protease-activated receptor-2, leading to widespread inflammation (Malamud et al., 2003). MBP also induces TGF-β release from mast cells. TGF-β1 and TGF-β3 were present in leukocytes found in active MS lesions (De Groot et al., 1999). Mast cell-derived IL-6 and TGF-β1 could participate in maturation/proliferation of Th17 cells, recently shown to be critical in MS and EAE (Weaver et al., 2006). IL-6 is upregulated in MS patients (Bongioanni et al., 2000b), and T cells from MS patients expressed significantly more IL-6 and TNF-α receptors compared with healthy controls (Bongioanni et al., 2000b). It is of interest that human mast cells release IL-6 selectively, without degranulation in response to IL-1 (Kandere-Grzybowska et al., 2003). Critical to mast cell involvement in MS is the mast cell's ability to secrete some mediators selectively without degranulation (Cao et al., 2005), as also suggested by ultrastructural observations in monkey demyelination (Letourneau et al., 2003).
We report here that MBP released high amounts of IL-8 from mast cells that are also stimulated to release IL-8 by Jurkat cells. IL-8 is elevated in serum and CSF of MS patients (Lund et al., 2004), and is a chemoattractant for neutrophils and monocytes triggering their adhesion to the endothelium (Salamon et al., 2005). Moreover, there is elevated expression of the IL-8 receptors CXCR1 and CXCR2 on macrophages, astrocytes, microglia and oligodendrocytes in MS lesions (Muller-Ladner et al., 1996). CSF IL-8 decreased when relapsing–remitting MS patients were treated with cladribine (Bartosik-Psujek et al., 2004); moreover, high doses of glucocorticoids decreased IL-8 production by monocytes in MS patients during relapse (Mirowska-Guzel et al., 2006). Interferon-β used to treat MS is also associated with the reduction of serum IL-8 levels (Lund et al., 2004).
There are no curative therapies presently available for MS (Fox and Ransohoff, 2004). Mast cells could serve as a therapeutic target for MS (Zappulla et al., 2002). The ability of the naturally occurring flavonoid luteolin to inhibit the processes described therein suggests that it may be useful in MS, alone or in combination with other treatments.