Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes

Abstract:  The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro , the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 m m . In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.     

Effects of prostaglandin E1 on human keratinocytes and dermal fibroblasts: A possible mechanism for the healing of skin ulcers

Abstract The effects of prostaglandin E, (PGE) on cell growth, cytokine production and interaction of cultured normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) were investigated. When NHKs were treated with PGE, directly, only a slight increase in cell growth and a transient decrease in interleukin 1 alpha (IL-lα) secretion were observed. No IL-6 was detected either before or after PGE, treatment. In addition, IL-8 and transforming growth factor alpha (TGFα) production were uninfluenced by PGE. The response of HDFs to PGE, differed from that of NHKs. Following PGE₁, treatment, IL-lα and TGFα. from HDFs remained undetectable while IL-6 production was enhanced markedly. IL-8 production was also slightly enhanced. Exposure of HDFs to PGE, for 96 hours significantly promoted cell proliferation. Two kinds of conditioned media (CM) were prepared by a brief feeding of HDFs with keratinocyte basic medium or Dulbecco's modified Eagle's medium supplemented with 5% PCS with or without PGE. NHKs proliferated more rapidly in CM than in corresponding basic medium. Moreover, CM prepared with PGE, treatment showed a stronger effect in promoting NHK proliferation than CM without PGE, treatment. This promoting effect was inhibited by anti-human IL-6 monoclonal antibody dose-dependently. These results indicate that fibroblasts are more sensitive than keratinocytes in response to PGE, and that, upon PGE, stimulation. HDF-derived IL-6 may play an essential role in NHK cell proliferation which may at least partly account for the beneficial effects of PGE, in the treatment of cutaneous liberations.     

Eicosapentaenoic acid, a n-3 polyunsaturated fatty acid differentially modulates TNF-alpha, IL-1alpha, IL-6 and PGE2 expression in UVB-irradiated human keratinocytes

In response to UVB-irradiation keratinocytes release a variety of cytokines and prostaglandins, including tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), interleukin 6 (IL-6), and prostaglandin E2 (PGE2). n-3 Polyunsaturated fatty acids (PUFA), mainly present in fish oil, can modulate cytokine synthesis, as predominantly studied in macrophages. In order to investigate the immune modulating actions of n-3 PUFA on the UVB response in human skin, we investigated the effect of eicosapentaenoic acid (EPA), a n-3 PUFA and a precursor of eicosanoid biosynthesis, on UVB-modulated TNF-alpha, IL-1alpha, IL-6, and PGE2 expression in normal human keratinocytes (NHK). We show that cultured NHK can efficiently take up EPA. Basal TNF-alpha expression is very low in NHK. IL-1alpha on the contrary is significantly present in untreated cultured NHK. Upon UVB-irradiation (32 mJ per cm2) TNF-alpha mRNA expression and secretion is induced and IL-1alpha mRNA expression is reduced, although IL-1alpha secretion is induced. EPA treatment results in higher TNF-alpha and IL-1alpha expression, both in nonirradiated and UVB-irradiated keratinocytes. Moreover EPA and UVB appear to act synergistically to superinduce TNF-alpha expression. EPA treatment results also in lipid peroxidation and in decreased PGE2 and IL-6 secretion after UVB-irradiation. In contrast to EPA, oleic acid (monounsaturated fatty acid) and linoleic acid (n-6 PUFA) treatment did not result in higher TNF-alpha or IL-1alpha levels in nonirradiated or UVB-irradiated NHK, indicating that the observed effects are specific for EPA. In conclusion, these results show that EPA can differentially modulate UVB-induced cytokine and prostaglandin synthesis in NHK.     

Human epidermal keratinocytes are a source of soluble ICAM-1 molecules.

A soluble form of the usually membrane-bound adhesion molecule ICAM-1 was detected in supernatants derived from human epidermal keratinocytes. Specifically, supernatants harvested from long-term cultured normal human keratinocytes, or from the spontaneously immortalized keratinocyte cell line HaCaT, did not contain significant amounts of sICAM-1, but shedding of sICAM-1 was found to be markedly induced upon stimulation of keratinocytes with rh IFN gamma. In contrast, cells from the two epidermoid carcinoma cell lines, KB and A431, constitutively shed significant amounts of sICAM-1 even without cytokine stimulation, and sICAM-1 contents in supernatants harvested from these cells were further increased upon stimulation of cells with rh IFN gamma. These studies indicate, that in addition to peripheral blood mononuclear cells and human melanoma cells, human epidermal keratinocytes constitute an important cellular source of sICAM-1. By binding to leukocyte LFA-1 molecules, keratinocyte-derived sICAM-1 may influence inflammatory responses in the skin. In addition, constitutive shedding of sICAM-1 by transformed human keratinocytes may represent a possible mechanism by which neoplastic keratinocytes escape from cytotoxicity.     

XBP1在白癜风皮损及应激HaCaT细胞中的表达

目的:检测XBP1在白癜风皮损组织及H2O2诱导的HaC aT细胞中的表达。方法:H2O2处理体外培养的HaC aT细胞。Real time PCR检测白癜风皮损中及应激HaC aT细胞中XBP1的表达;ELISA检测应激HaC aT细胞中特异性抑制剂抑制XBP1活化前后IL-6和IL-8的表达。结果:白癜风皮损和应激HaC aT细胞中XBP1 mRNA显著高于正常对照;应激HaC aT细胞中IL-6和IL-8水平高于空白对照组,抑制XBP1活化后两者分泌减少。结论:XBP1在白癜风皮损组织中显著上调,促进应激的HaC aT细胞分泌IL-6、IL-8。     

Effects of Etretinate on Keratinocyte Proliferation and Secretion of Interleukin-1α (IL-1α) and IL-8

Etretinate has proven to be effective in the treatment of psoriasis. Since abnormal proliferation and cytokine secretion are well-known features of psoriatic epidermis, we studied the in vitro effects of etretinate on these two processes using human keratinocytes. Etretinate promoted proliferation of normal human keratinocytes (NHKs) grown in keratinocyte growth medium (KGM) but not in growth factor-deficient keratinocyte basic medium (KBM). Moreover, etretinate partly overcame growth inhibition by PMA. Etretinate was shown to have an effect on either IL-1α or IL-8 secretion in unstimulated NHKs. In HSC-1, a human squamous cell carcinoma cell line cultured in 20% FCS/DMEM, inhibited IL-1α secretion and enhanced IL-8 secretion. These results indicate that the effects of etretinate on keratinocyte proliferation and cytokine secretion may depend on cell type and culture conditions. Stimulation of NHKs with PMA significantly enhanced IL-1α and IL-8 secretion, and these effects were inhibited by etretinate. However, etretinate failed to inhibit rTNFα-induced IL-8 secretion, suggesting that etretinate regulation of NHK cytokine secretion may also depend on the stimulus. As treatment of keratinocytes or epidermis with PMA can induce psoriasis-like changes, so might the experimental “anti-PMA” activity of etretinate be related to its therapeutic benefit in the treatment of psoriasis.     

Transient receptor potential vanilloid-1 mediates heat-shock-induced matrix metalloproteinase-1 expression in human epidermal keratinocytes

Transient receptor potential vanilloid-1 (TRPV1), a heat-gated channel, was recently found on human keratinocytes and the activation of epidermal TRPV1 was known to induce release of proinflammatory mediators. However, the functional consequences of TRPV1 activation in cutaneous physiology and pathology have not been elucidated clearly. In this study, we investigated the role of TRPV1 on the matrix metalloproteinase (MMP)-1 expression induced by heat shock in human epidermal keratinocytes. Heat shock induced the expression of MMP-1 mRNA and protein in a temperature-dependent manner in an immortalized human keratinocyte cell line (HaCaT) and normal human epidermal keratinocytes (NHK). Heat-shock-induced MMP-1 expression was decreased by treatment of the TRPV1 inhibitors (capsazepine and ruthenium red) or knockdown of TRPV1 using RNA interference in HaCaT cells. Overexpression of TRPV1 greatly increased heat-shock-induced MMP-1 promoter activity in HEK 293 cells. Furthermore, direct activation of TRPV1 by capsaicin, a TRPV1 agonist, increased MMP-1 expression. We found that heat shock induced calcium influx through TRPV1 and that extracellular calcium was necessary for heat-shock-induced MMP-1 expression in HaCaT cells. Taken together, our results suggest that heat-shock-induced MMP-1 expression is mediated by activation of TRPV1 and is dependent on a calcium-dependent signaling process in human epidermal keratinocytes.     

TNF receptor I on human keratinocytes is a binding partner for staphylococcal protein A resulting in the activation of NF kappa B, AP-1, and downstream gene transcription

Primary human keratinocytes and immortalized HaCaT cells were analysed for their capacity to bind purified staphylococcal protein A (SpA). Co-incubation with FITC-labelled SpA led to a dose-depending attachment. Pull-down experiments with cellular extracts revealed the TNFα receptor I (TNF RI) as binding partner on keratinocytes. Thus, we next looked for expression of this receptor in human epidermis and cultured keratinocytes. TNF RI is strongly expressed on all keratinocytes analysed, both at the mRNA and protein level and activation by SpA at optimal doses of 50-100 μg/ml resulted in the phosphorylation of the TNF RI downstream kinases MEK1/2, JNK1/2, and p38 subsequently leading to translocation of the p65 NF kappa B subunit and AP-1 into the nucleus. This translocation was then followed by increased expression of IL-8 and COX-2, two known NF kappa B-induced pro-inflammatory genes. To further test the relevance of our findings, we analysed in vitro production of over 100 strains isolated from atopic eczema showing that more than 85% of the tested strains produced extracellular SpA in substantial amounts. Thus, besides superantigens, haemolysins, and other cell wall components, Staphylococcus aureus exerts pro-inflammatory stimuli on human keratinocytes through the production of SpA signalling through TNF RI.     

The participation of proliferative keratinocytes in the preimmune response to sensitizing agents

The aims of this study were to investigate whether keratinocytes are capable of playing a direct preimmune role in the pathophysiology of allergic contact dermatitis (ACD) and to examine to what extent the degree of differentiation might influence this. We measured the ability of sensitizing agents to up-regulate intercellular adhesion molecule 1 (ICAM-1) expression in cultured normal human keratinocytes (NHK) and in the transformed human keratinocyte HaCaT cell line. In proliferative HaCaT cells, following a 24 h exposure, nickel compounds, para-phenylenediamine (pPD) and 1-chloro-2,4-dinitrobenzene produced a concentration-dependent up-regulation of ICAM-1 expression without reducing cell viability, while K₂Cr₂O₇ led to ICAM-1 up-regulation at cytotoxic concentrations, and CrCl₃ was without effect. In NHK, NiSO₄ and pPD induced ICAM-1 expression to a significantly greater extent in proliferative cells than in differentiated cells, where involucrin expression was measured to assess the differentiation state. NiSO₄- or pPD-pretreatment of proliferative HaCaT cells enhanced T-cell binding, which was abolished by neutralizing antibodies to ICAM-1 or CD18. Our investigations concerning the involvement of oxidative stress in the induction of ICAM-1 expression in response to sensitizing agents were inconclusive. The oxidizing agents FeCl₃ and H₂O₂ up-regulated ICAM-1 expression in HaCaT cells but there was no clear relationship between the ability of agents to induce ICAM-1 expression and their ability to alter the levels of reduced glutathione. Although pPD increased interleukin-1α release from NHK, this cytokine was not capable of inducing ICAM-1 expression in NHK. Tumour necrosis factor-α, which does induce ICAM-1 expression in NHK, was not detected in response to pPD, arguing against an autocrine pathway of ICAM-1 induction in response to pPD. In summary, we report the direct interaction of sensitizing agents with keratinocytes leading to the generation of immune signals, particularly by proliferative keratinocytes, suggesting an active role for the proliferative keratinocyte in the pathophysiology of ACD.     

Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro

BACKGROUND: thymus and activation-regulated chemokine (TARC)/CCL17 is a CC chemokine that selectively attracts Th2-type lymphocytes. Immunohistochemical analyses have revealed that TARC is expressed in the epidermal keratinocytes of atopic dermatitis (AD), suggesting TARC involvement in the pathogenesis of the disease. However, keratinocyte TARC production has been described only in the transformed keratinocyte cell line HaCaT. OBJECTIVE: to examine TARC production in normal human epidermal keratinocytes (NHEK) in vitro. METHODS: the expression of TARC mRNA and protein were examined in NHEK and HaCaT cells stimulated with various cytokines. RESULTS: stimulation with inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha failed to induce TARC mRNA expression in NHEK. However, stimulation with IFN-gamma and TNF-alpha together enhanced expression slightly. ELISA analysis failed to detect TARC protein in NHEK culture supernatant, even following stimulation with IFN-gamma and TNF-alpha. In contrast, HaCaT cells produced TARC protein even without stimulation of cytokines. CONCLUSION: these results indicate that production of TARC by HaCaT cells is a phenomenon specific to the cell line and the observation on TARC in HaCaT cells can not be generalized. NHEK do not produce TARC protein in vitro.     

Serum factors regulate the expression of the proliferation-related genes α5 integrin and keratin 1, but not keratin 10, in HaCaT keratinocytes

Abstract In the highly coordinated programme of gene expression during keratinocyte proliferation and differentiation, ·5 integrin and keratins 1 and 10 (K1/K10) may play important regulatory roles. We were interested in seeing whether, in continuously growing, immortalized HaCaT keratinocytes, similar to normal keratinocytes, the expression of ·5 integrin and K1/K10 was related to cell proliferation and differentiation. After release from cell quiescence the expression of ·5 integrin, both at the mRNA and protein levels, was upregulated in the cells. At the same time, K1/K10 mRNA and protein expression decreased dramatically, while the mRNA for D1 cyclin became detectable, and the cells became highly proliferative. These findings indicate that ·5 integrin and K1/K10 are involved in the regulation of HaCaT proliferation and differentiation, as in normal keratinocytes. However, HaCaT cells are different from normal keratinocytes in their ability to lose K1/K10 expression. There is no evidence that the expression of K1/K10 can be reversed in normal keratinocytes. This ability of dedifferentiation might be a unique feature of HaCaT cells and may be a key component of their immortalized nature. We also found that serum factors regulate mRNA expression of ·5 integrin and K1, but not of K10, in HaCaT cells. This information could be relevant to the understanding of normal epidermal differentiation. Received: 28 August 2000 / Revised: 27 October 2000 / Accepted: 3 February 2001     

角质形成细胞μ-阿片受体表达的研究

目的：研究不同特性的角质形成细胞μ-阿片受体的表达情况。方法：以体外培养的角质形成细胞株HaCaT细胞、人鳞状细胞癌(简称鳞癌)细胞株A431、神经母细胞瘤SK-N-SH细胞株为对象，采用逆转录(RT)-PCR方法研究细胞μ-阿片受体的表达。结果：在常规体外培养条件下，角质形成细胞株HaCaT细胞、人鳞癌细胞株A431的RT-PCR结果显示有μ-阿片受体mRNA的表达，后者的表达水平略高于前者。结论：μ-阿片受体在角质形成细胞的表达，为神经系统和皮肤通过神经肽直接发生作用提供依据。     

Cell migration and MMP-9 secretion are increased by epidermal growth factor in HaCa T-ras transfected cells

Abstract: Mutated RAS oncoproteins and epidermal growth factor (EGF) are thought to contribute to the proliferative, invasive and metastatic properties of transformed cells. In the present study, we investigated the role of EGF in two H- ras transfected clones and compared it to that in the parental cell line, HaCaT and primary cultured keratinocytes. Our findings show that the motility on type I collagen, measured by the migration index, was similar for both the HaCaT cell line and normal human keratinocytes, whereas it was higher for the HaCaT- ras clones. These results suggest an involvement of the ras oncogene in the stimulation of cell migration. EGF, in cell pretreatment or during the migration assay also caused an increase in migration of all the cells, but preserved the difference between HaCaT and HaCaT- ras . However, no significant difference in EGF-R expression was detected between normal cultured keratinocytes, HaCaT and HaCaT- ras cell lines with or without EGF pretreatment. Moreover, when the cells were stimulated with EGF, the MMP-9 activity was greatly increased in a dose-dependent manner in all the cells, and EGF stimulation particularly highlights the increased amount of MMP-9 in HaCaT- ras cells compared to HaCaT cells. In conclusion, EGF is able to enhance motility and to up-regulate MMP-9 activity in all cells, but with a higher impact in HaCaT- ras cells without an overexpression of EGF-R. As EGF acts in synergy with the H- ras mutation, they could be implicated in the local invasion by the HaCaT- ras clones.     

A comparative study of UV-induced cell signalling pathways in human keratinocyte-derived cell lines

Ultraviolet (UV) radiation can activate the p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK) and nuclear factor-κB (NFκB) pathways in skin cells. HaCaT cells are widely used as a primary keratinocyte substitute to study these pathways. However, like most squamous cell carcinomas (SCCs), it contains a dysfunctional p53. It is unclear if HaCaT cells activate these signalling pathways similarly to SCC cells (Colo16) or to primary human epidermal keratinocytes (HEK). In this study, the UV activation (UVA, UVB, UVA + B, UVB + A) of p38 MAPK, JNK and NFκB pathways, and TNFα secretion by HEK, HaCaT and Colo16 cells were investigated. The signalling pathway activation was UV-type and dose-dependent with UVB + A radiation inducing a high p38 and JNK activation. HaCaT cells exhibited 2- to 4-fold higher activity of the p38 (771 % at 60 min) and JNK (794 % at 30 min) pathways following UVB + A radiation than did HEK cells (p38: 367 % at 15 min and JNK: 184 % at 30 min). While both HaCaT and Colo16 cells did not activate the NFκB pathway, Colo16 cells had a lower p38 and higher JNK activity than HaCaT cells. Irradiated HaCaT cells produced less TNFα (UVB: 3.5 pg/ml), while HEK cells produced the most (UVB: 1,296 pg/ml). When co-exposed to IL1α, irradiated HaCaT had the greatest fold of TNFα release (UVB: 16.2-fold, UVA + B: 8.9-fold and UVB + A: 6.1-fold). The pattern of activation and TNFα secretion of HaCaT cells mirrored that of Colo16 cells. It is likely that the presence of molecular alterations in HaCaT cells may be responsible for its different responses to that seen for HEK cells. The results of this study suggest caution in using HaCaT cells as a substitute for normal keratinocytes in investigating UV-induced cells signalling pathways.     

Increased release of the tumour necrosis factor receptor p75 by immortalized human keratinocytes results from an activated shedding mechanism and is not related to augmented steady-state levels of p75 mRNA

The soluble tumour necrosis factor receptor I (sTNFRI, p55) is produced at similar levels by both immortalized (A431, HaCaT, KB) and primary normal human keratinocytes (HNK), whereas the soluble TNFR II (sTNFR II, p75) appears to be specifically released only by immortalized human keratinocytes. The purpose of this study was to investigate whether the increase in p75 secretion by immortalized human keratinocytes is due to an increased shedding of the receptor from the cell membrane, or is related to increased steady-state levels of p75 mRNA. FACS analysis showed that levels of membranous p75 decreased in a time-dependent manner in immortalized cells cultured for 1, 3, 6, 12 and 24 h, while remaining unchanged in HNK throughout. Northern blot analysis showed that after 12 h of culture, when p75 expression was decreased on the cell membrane of all immortalized cells, there was no significant difference in steady state levels of p75 mRNA between immortalized keratinocytes and HNK. Supernatants of immortalized cells, cultured for 24 h contained distinct levels of p75, while levels of p75 in supernatants of HNK were under the detection limit, confirming that the p75 decrease on the cell membrane results from increased p75 shedding from the cell membrane of immortalized cells. In contrast to p75, p55 was continuously expressed on the cell membrane of normal and immortalized keratinocytes without significant variation throughout the entire 24-h culture period and was similarly shed by both cell types. These results suggest that immortalized keratinocytes are specifically activated for shedding of p75 from the cell membrane. Since p75 has a high affinity for TNF, the release of this receptor may imply a direct role in the escape of malignant/transformed keratinocytes from the TNF-mediated immune response. Received: 25 January 1996     

Semaphorin 7A on keratinocytes induces interleukin-8 production by monocytes

Background

Semaphorin 7A (Sema7A) expressed on activated T cells stimulates cytokine production in monocytes through its receptor, α1β1 integrin.

Objective

To study the significance of Sema7A expressed on keratinocytes in skin inflammation where interaction between keratinocytes and β1-integrin expressing inflammatory cells, such as monocytes, takes place.

Methods

The regulation of Sema7A expression on keratinocytes by various cytokines was studied by flow cytometry and immunoblot. β1-integrin expressing human monocyte cell line, THP-1 cells, were co-cultured with paraformaldehyde-fixed normal human epidermal keratinocytes (NHK) and IL-8 production by THP-1 cells was studied. The significance of β1-integrin or Sema7A within this cell interaction was examined by the experiments using β1-integrin blocking antibody or Sema7A siRNA.

Results

IFN-γ and TNF-α slightly increased Sema7A expression, while IL-4 decreased it. Among cytokines tested, TGF-β1 most strikingly increased the Sema7A expression on NHK. When NHK was stimulated by TGF-β1, paraformaldehyde-fixed, and co-cultured with THP-1 cells, IL-8 production by THP-1 cells was increased compared to THP-1 cells only. When THP-1 cells were pretreated with β1-integrin blocking antibody, this increase in IL-8 production by THP-1 cells was inhibited. Likewise, when NHK were pretreated with Sema7A siRNA before fixation and co-cultured with THP-1 cells, increase in IL-8 production by THP-1 cells was inhibited.

Conclusion

Our results suggest that Sema7A on keratinocytes and β1-integrin on monocytes contribute to monocyte activation by keratinocytes within skin inflammation, such as psoriasis or wound.     

A GPR40 agonist GW9508 suppresses CCL5, CCL17, and CXCL10 induction in keratinocytes and attenuates cutaneous immune inflammation

G-protein coupled receptors (GPCR) exert diverse physiological functions, many of which are exploited therapeutically. The roles of GPCR in keratinocytes in immune response in the skin, however, remain poorly defined. In this study, we focused on Gi-coupled GPCR in keratinocytes and defined their actions in immunoactivation of cultured keratinocytes in vitro and immune reaction in the skin in vivo. We first activated HaCaT cells by tumor necrosis factor (TNF)-α and IFN-γ and examined effects of various ligands for GPCR on production of CCL17 and CCL5. Agonists for Gi-coupled receptors, particularly GW9508 for GPR40, inhibited CCL17 and CCL5 expression in a pertussis toxin-sensitive manner. The inhibitory effect by GW9508 was abrogated by depletion of GPR40 with RNA interference. GW9508 further suppressed expression of IL-11, IL-24, and IL-33 induced in HaCaT cells by TNF-α and IFN-γ. GW9508 also inhibited CCL5 and CXCL10 production by normal human epidermal keratinocytes. Administration of GW9508 topically to the skin in the challenging phase suppressed ear swelling in a repeated hapten application model and contact hypersensitivity with downregulation of CCL5 and CXCL10, respectively. Thus, in the skin, stimulation of Gi-coupled receptors attenuates induction of critical cytokines and chemokines by proinflammatory cytokines in keratinocytes and suppresses allergic inflammation in the skin.