Human intestinal mast cells are capable of producing different cytokine profiles: role of IgE receptor cross-linking and IL-4

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-alpha. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.     

Primary administration of Lactobacillus johnsonii NCC533 in weaning period suppresses the elevation of proinflammatory cytokines and CD86 gene expressions in skin lesions in NC/Nga mice

The administration of probiotic lactic acid bacteria (LAB) has been studied for its potential to prevent atopic dermatitis (AD). The objective of this study was to assess the inhibitory mechanism of a skin lesion by LAB using an experimental model that we previously demonstrated in NC/Nga mice. Lactobacillus johnsonii NCC533 (La1) was administered orally to the La1 group from 20 to 22 days after birth, while phosphate-buffered saline was given to the control group. After the induction of skin lesions in 6-week-old mice, the expression of genes supposedly involved in AD was evaluated. Gene expression of the proinflammatory cytokines [interleukin-8 (IL-8), IL-12 and IL-23] was significantly enhanced in the lesional skin of the control group by the induction of the lesion, whereas gene expression of those in the La1 group was not elevated. Interestingly, expression of the costimulatory molecule CD86 showed a pattern similar to the expression of the cytokines in the lesional skin. Moreover, the La1 group showed a significantly lower gene expression of CD86 in Peyer's patches and mesenteric lymph nodes than the control group. The suppression of proinflammatory cytokines and CD86 by primary administration of La1 may significantly contribute to the inhibitory effect on the skin lesion.     

Cutting Edge: Proinflammatory and Th2 cytokines synergize to induce thymic stromal lymphopoietin production by human skin keratinocytes

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells (DC) and can initiate allergic inflammation. The factors inducing the production of human TSLP are not known. In this study, we show that proinflammatory (TNF-alpha or IL-1alpha) and Th2 (IL-4 or IL-13) cytokines synergized to induce the production of TSLP in human skin explants. TSLP production in situ was restricted to epidermal keratinocytes of the suprabasal layer. TSLP production could not be inhibited by factors regulating Th2 inflammation, such as IL-10, TGF-beta, or IFN-gamma. Cytokine-treated skin culture supernatants induced the maturation of blood CD11c(+) DC in a TSLP-dependent manner. Our data provide the first evidence of TSLP induction and subsequent DC activation in human skin. Blocking TSLP-inducing cytokines could represent a novel strategy for the treatment of allergic diseases.     

Human cord blood-derived mast cells synthesize and release I-309 in response to IgE

Mast cells are the central mediating cells of allergic reactions. Binding of allergen specific IgE to high affinity IgE receptor (Fcepsilon RI) and subsequent binding of allergen by the IgE causes receptor cross-linking and activation. In a study examining the differential gene expression in human cord blood-derived mast cells (CBMCs) mediated by activation of Fcepsilon RI both with IgE and IgE followed by cross-linking with alpha-IgE, the chemokine I-309 was found to be upregulated. I-309 is the ligand for the CCR8 receptor and is responsible for chemoattraction of TH2 type T-cells. Interestingly, I-309 RNA and protein levels were elevated not only in response to IgE/alpha-IgE activation but also by IgE alone. In addition, the I-309 levels were augmented by growth of the CBMCs in the presence of the proinflammatory cytokine IL-4. GM-CSF and MIP-1alpha secretion was also induced by IgE. These results suggest that IgE, through the production and release of cytokines such as I-309, GM-CSF and MIP-1alpha could promote an inflammatory reaction in the absence of antigen stimulation of mast cells.     

Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines

Homeostatic regulation of epidermal keratinocytes is controlled by the local cytokine milieu. However, a role for suppressor of cytokine signaling (SOCS), a negative feedback regulator of cytokine networks, in skin homeostasis remains unclear. Keratinocyte specific deletion of Socs3 (Socs3 cKO) caused severe skin inflammation with hyper-production of IgE, epidermal hyperplasia, and S100A8/9 expression, although Socs1 deletion caused no inflammation. The inflamed skin showed constitutive STAT3 activation and up-regulation of IL-6 and IL-20 receptor (IL-20R) related cytokines, IL-19, IL-20 and IL-24. Disease development was rescued by deletion of the Il6 gene, but not by the deletion of Il23, Il4r, or Rag1 genes. The expression of IL-6 in Socs3 cKO keratinocytes increased expression of IL-20R-related cytokines that further facilitated STAT3 hyperactivation, epidermal hyperplasia and neutrophilia. These results demonstrate that skin homeostasis is strictly regulated by the IL-6-STAT3-SOCS3 axis. Moreover, the SOCS3-mediated negative feedback loop in keratinocytes has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3.     

Dysregulation of melanocyte function by Th17-related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris

The aim of this study was to determine whether CD4(+) IL-17A(+) Th17 cells infiltrate vitiligo skin and to investigate whether the proinflammatory cytokines related to Th17 cell influence melanocyte enzymatic activity and cell fate. An immunohistochemical analysis showed Th17 cell infiltration in 21 of 23 vitiligo skin samples in addition to CD8(+) cells on the reticular dermis. An in vitro analysis showed that the expression of MITF and downstream genes was downregulated in melanocytes by treatment with interleukin (IL)-17A, IL-1β, IL-6, and tumor necrosis factor (TNF)-α. Treatment with these cytokines also induced morphological shrinking in melanocytes, resulting in decreased melanin production. In terms of local cytokine network in the skin, IL-17A dramatically induced IL-1β, IL-6, and TNF-α production in skin-resident cells such as keratinocytes and fibroblasts. Our results provide evidence of the influence of a complex Th17 cell-related cytokine environment in local depigmentation in addition to CD8(+) cell-mediated melanocyte destruction in autoimmune vitiligo.     

In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-gamma-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-gamma production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-gamma-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-gamma, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.     

CD57 Expression and Cytokine Production by T Cells in Lesional and Unaffected Skin from Patients with Psoriasis

Background

The immunopathogenic mechanisms leading to psoriasis remain unresolved. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and the proportion of CD57 expressing CD8+ T cells is increased in a number of inflammatory conditions.

Methodology

We examined the expression of CD57 on T cells in the skin of patients affected with psoriasis, comparing lesional and unaffected skin. We also assessed functionality of the T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-gamma, IL-2, IL-33, TNF-alpha, IL-21, IL-22, and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin biopsies of psoriasis patients.

Principal Findings

We observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly higher in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriatic lesional skin produced higher levels of IL-17A, IL-22, and IFN-gamma compared to unaffected skin, while sorted CD8+ T cells from lesional skin produced higher levels of IL-17, IL-22, IFN-gamma, TNF-alpha, and IL-2 compared to unaffected skin.

Conclusions/Significance

These findings suggest that T cells in unaffected skin from psoriasis patients exhibit a phenotype compatible with replicative inability. As they have a lower replicative capacity, CD57+ T cells are less frequent in lesional tissue due to the high cellular turnover.     

New insights into the pathogenesis of vitiligo: imbalance of epidermal cytokines at sites of lesions

Vitiligo is a skin disease that is caused by selective destruction of melanocytes and is characterized by white spots. Melanocytes and keratinocytes seem to exhibit a functional close relationship, mediated at least in part by keratinocyte-derived cytokines, which seem important for survival and activity of melanocytic cells. We wanted to investigate the hypothesis that in vitiligo the expression of epidermal cytokines may be modified compared with normal skin. In 15 patients with active, non-segmental vitiligo, biopsies were obtained from lesional, perilesional and non-lesional skin; normal skin from five healthy donors was also tested. Tissue sections were tested using immunohistochemistry for the expression of keratinocyte-derived cytokines with stimulating activity, such as granulocyte-monocyte colony stimulating factor (GM-CSF), basic fibroblastic growth factor (bFGF), and stem cell factor (SCF) or with inhibiting activity, such as interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) on melanocytes. Cytokine receptors and specific melanocytic markers were also investigated. No melanocyte was identified in lesional skin by means of specific markers or c-kit receptor, whereas in perilesional, non-lesional and healthy skin, melanocytes were found in similar number. In vitiligo skin a significantly lower expression of GM-CSF, bFGF and SCF was found, and a significantly higher expression of IL-6 and TNF-alpha was detected, compared with perilesional, non-lesional and healthy skin. In conclusion, we provided evidence that a significant change of epidermal cytokines exists in vitiligo skin compared with perilesional, non-lesional and healthy skin, suggesting that the cytokine production of epidermal microenvironment may be involved in vitiligo.     

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.     

Cbl-b is a negative regulator of inflammatory cytokines produced by IgE-activated mast cells

c-Cbl and Cbl-b E3 ubiquitin ligases are abundantly expressed in hemopoietic cells where they negatively regulate the activity and levels of many cell surface receptors and associated signaling molecules. By comparing bone marrow-derived mast cells from c-Cbl and Cbl-b-deficient mice it has recently been shown that Cbl-b is the dominant family member for negatively regulating signaling responses from high-affinity IgE receptors. In this study, we suggest that a possible reason for the greater enhancement of IgE receptor signaling in Cbl-b-deficient mice is the relatively higher levels of Cbl-b protein over c-Cbl in mast cells compared with other hemopoietic cells. We also directly compare mast cells from c-Cbl and Cbl-b-deficient mice and find that loss of Cbl-b, but not c-Cbl, increases cell growth, retards receptor internalization, and causes the sustained tyrosine phosphorylation of Syk and its substrates. However, loss of Cbl-b does not enhance the activation of ERK or Akt, nor does it promote a greater calcium response. Furthermore, loss of Cbl-b or c-Cbl does not increase levels of the Syk or Lyn protein tyrosine kinases. Most notable, however, is the extremely large increase in the production of proinflammatory cytokines TNF-alpha, IL-6, and MCP-1 by Cbl-b(-/-) mast cells compared with levels produced by c-Cbl(-/-) or wild-type cells. This marked induction, which appears to be restricted to these three cytokines, is dependent on IgE receptor activation and correlates with enhanced IkappaB kinase phosphorylation. Thus, Cbl-b functions as a potent negative regulator of cytokines that promote allergic and inflammatory reactions.     

In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9

Mast cells can play detrimental roles in the pathophysiology and mortality observed in anaphylaxis and other Th2-dominated allergic diseases. In contrast, these cells contribute to protective host defense mechanisms against parasitic worm infections. After IgE/Ag activation, mast cells can produce multiple cytokines that may enhance allergic inflammations, while a similar panel of Th2-related cytokines may support immunological strategies against parasites. Here we report that in primary mouse bone marrow-derived mast cells activated by ionomycin or IgE/Ag, the proinflammatory mediator IL-1 (alpha or beta) up-regulated production of IL-3, IL-5, IL-6, and IL-9 as well as TNF, i.e., cytokines implicated in many inflammatory processes including those associated with allergies and helminthic infections. IL-1 did not induce significant cytokine release in the absence of ionomycin or IgE/Ag, suggesting that Ca-dependent signaling was required. IL-1-mediated enhancement of cytokine expression was confirmed at the mRNA level by Northern blot and/or RT-PCR analysis. Our study reveals a role for IL-1 in the up-regulation of multiple mast cell-derived cytokines. Moreover, we identify mast cells as a novel source of IL-9. These results are of particular importance in the light of recent reports that strongly support a central role of IL-9 in allergic lung inflammation and in host defense against worm infections.     

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.     

Mapping of the CD23 Binding Site on Immunoglobulin E (IgE) and Allosteric Control of the IgE-Fc{epsilon}RI Interaction

IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcεRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcεRI, whereas IgE-CD23 interactions control IgE expression levels. We have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. We show that the CD23 and FcεRI interaction sites are at opposite ends of the Cε3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FcεRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE-FcεRI and IgE-CD23 interactions.     

Cytokine production by skin-derived mast cells: endogenous proteases are responsible for degradation of cytokines

The current study characterizes the cytokine protein (ELISA) and mRNA (gene array and RT-PCR) profiles of skin-derived mast cells cultured under serum-free conditions when activated by cross-linking of Fc epsilonRI. Prior to mast cell activation, mRNA only for TNF-alpha was detected, while after activation mRNA for IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF substantially increased, and for IL-4 it minimally increased. However, at the protein level certain recombinant cytokines, as measured by ELISAs, were degraded by proteases released by these skin-derived mast cells. IL-6 and IL-13 were most susceptible, followed by IL-5 and TNF-alpha; GM-CSF was completely resistant. These observations also held for the endogenous cytokines produced by activated mast cells. By using protease inhibitors, chymase and cathepsin G, not tryptase, were identified in the mast cell releasates as the likely culprits that digest these cytokines. Their cytokine-degrading capabilities were confirmed with purified chymase and cathepsin G. Soy bean trypsin inhibitor, when added to mast cell releasates, prevented the degradation of exogenously added cytokines and, when added to mast cells prior to their activation, prevented degradation of susceptible endogenous cytokines without affecting either degranulation or GM-CSF production. Consequently, substantial levels of IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF were detected 24-48 h after mast cells had been activated, while none were detected 15 min after activation, by which time preformed granule mediators had been released. IL-4 was not detected at any time point. Thus, unless cytokines are protected from degradation by endogenous proteases, cytokine production by human mast cells with chymase and cathepsin G cells may be grossly underestimated.     

IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines

IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, IL-10, and IL-17 to modulate IP-10, Mig, and I-TAC in cultured human keratinocytes and CXCR3 expression in T cells from allergic contact dermatitis (ACD). IL-4, but not IL-10 or IL-17, significantly up-regulated IFN-gamma- or TNF-alpha-induced IP-10, Mig, and I-TAC mRNA accumulation in keratinocytes and increased the levels of IP-10 and Mig in keratinocyte supernatants. Immunohistochemistry of skin affected by ACD revealed that >70% of infiltrating cells were reactive for CXCR3 and that CXCR3 staining colocalized in CD4+ and CD8+ T cells. Nickel-specific CD4+ and CD8+ T cell lines established from ACD skin produced IFN-gamma and IL-4 and expressed moderate to high levels of CXCR3. Finally, CXCR3 agonistic chemokines released by stimulated keratinocytes triggered calcium mobilization in skin-derived nickel-specific CD4+ T cells and promoted their migration, with supernatant from keratinocyte cultures stimulated with IFN-gamma and IL-4 attracting more efficaciously than supernatant from keratinocytes activated with IFN-gamma alone. In conclusion, IL-4 exerts a proinflammatory function on keratinocytes by potentiating IFN-gamma and TNF-alpha induction of IP-10, Mig, and I-TAC, which in turn may determine a prominent recruitment of CXCR3+ T lymphocytes at inflammatory reaction sites.