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BASIC RESEARCH
Year : 2009  |  Volume : 54  |  Issue : 3  |  Page : 237-239
Cell proliferation and cytokine induction by TNF-α of psoriatic keratinocytes are not different from normal keratinocytes in vitro


Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaokahigashi, Asahikawa 078-8510, Japan

Date of Web Publication10-Sep-2009

Correspondence Address:
Hidetoshi Takahashi
Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaokahigashi, Asahikawa 078-8510
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-5154.55631

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   Abstract 

Background: Recent studies indicate that various cytokines including tumor necrosis factor-á (TNF-á) play an essential role in the induction and maintenance of psoriatic lesion. Aims: To compare the cell proliferation of keratinocytes by various cytokines and TNF-á-induced cytokine secretion among normal keratinocytes, uninvolved, and involved keratinocytes. Methods: The keratinocytes from normal skin, uninvolved, and involved psoriasis were cultured in the presence of IL-6, IL-8, epidermal growth factor (EGF), hepatocyte growth factor (HGF), transforming growth factor-α (TGF-α) epiregulin, amphiregulin, and TNF-α and then MTT assay for keratinocytes proliferation was performed. Furthermore, TNF-α-induced secretion of IL-6, IL-8, EGF, HGF, TGF-α, epiregulin, and amphiregulin were compared among these keratinocytes. Results: IL-6, IL-8, EFG, TGF-á, epiregulin, and amphiregulin, but not TNF-α increased keratinocyte proliferation of normal, psoriatic uninvolved, and involved skin. The increased cell proliferation by these cytokines and growth factors were not different among the keratinocytes derived from normal skin, uninvolved, and involved psoriasis. The significant induction of TNF-α increased IL-6, IL-8, EGF, HGF, TGF-α, epiregulin, and amphiregulin, but the increase in these cytokines and growth factors were not different among normal skin, uninvolved, and involved psoriasis. Conclusion: Cell proliferation by various cytokines and growth factors and TNF-α-induced cytokine secretion are not different between normal and psoriatic keratinocytes. α


Keywords: Cell proliferation, psoriasis, TNF-α


How to cite this article:
Takahashi H, Tsuji H, Hashimoto Y, Ishida-Yamamoto A, Iizuka H. Cell proliferation and cytokine induction by TNF-α of psoriatic keratinocytes are not different from normal keratinocytes in vitro. Indian J Dermatol 2009;54:237-9

How to cite this URL:
Takahashi H, Tsuji H, Hashimoto Y, Ishida-Yamamoto A, Iizuka H. Cell proliferation and cytokine induction by TNF-α of psoriatic keratinocytes are not different from normal keratinocytes in vitro. Indian J Dermatol [serial online] 2009 [cited 2019 Sep 23];54:237-9. Available from: http://www.e-ijd.org/text.asp?2009/54/3/237/55631



   Introduction Top


Psoriasis has been characterized by hyperproliferation accompanied by acanthosis and aberrant differentiation of keratinocytes. Although the precise mechanism of hyperproliferation in psoriasis is still unclear, several growth factors and cytokines, such as epidermal growth factor (EGF), amphiregulin, epiregulin, IL-6, and IL-8, are assumed to be important. [1] Furthermore, it is supposed that TNF-α and INF-α, which are derived from dendritic cells and T cells, regulate the expression of these growth factors and cytokines. Because cyclosporine and anti-TNF-α agents, such as infliximab, adalimumab, and eternercept, are effective for psoriasis, [2] the trigger of keratinocytes hyperproliferation is presumed to be cellular immune response mediated by T cells and dendritic cells.[3] However, it is still unclear whether the keratinocytes derived from psoriasis are inherently distinct from normal keratinocytes. In the present study, we compared cell proliferation induced by various cytokines and growth factors as well as TNF-α-induced secretion of cytokine/growth factors among keratinocytes from normal skin, uninvolved, and involved psoriasis.


   Materials and Methods Top


Patients

Skin samples were collected from five psoriasis (36 to 55 years old) patients and five healthy individuals (28 to 48 years old) who underwent plastic surgery. All patients had untreated, moderate to severe plaque type psoriasis. The skin was obtained after informed consent and with the approval of the Ethical Committee of the Asahikawa Medical College.

Cell culture

Primary keratinocytes were obtained from normal, psoriatic involved, and uninvolved skin by minor modifications of the methods of Nickoloff et al . [4] Briefly, dermal-epidermal separation was achieved by incubation in Dulbecco's-modified Eagle's minimal essential medium supplemented with 1% dispase. The epidermis was treated with 0.25% trypsin/0.02% EDTA for 15min at 37°C. Then the keratinocytes were cultured in keratinocytes growth medium (KGM) containing insulin (5μg/ml), and bovine pituitary extract (50μg/ml) at 37°C in air containing 5% CO 2 . The calcium concentration in the medium was 0.1 mM. The keratinocytes were maintained in a subconfluent state by subculturing in every 4-5 days and various assays were performed.

MTT assay for keratinocytes proliferation

The primary keratinocytes obtained were cultured in 96-well microtiter plates (1 × 10 3 cells/well), and incubated for 24 h with the indicated concentrations of cytokines: TNF-α (1ng/ml), EGF (10ng/ml), amphiregulin (1ng/ml), hepatocyte growth factor (HGF) (10ng/ml), epiregulin (10ng/ml), transforming growth factor-α (TGF-α) (1ng/ml), IL-6 (0.5ng/ml), and IL-8 (0.5ng/ml)). These concentrations were chosen to obtain the maximal effect on keratinocyte cell proliferation (data not shown). Non-radioactive MTT proliferation assay using tetrazolium was performed according to the protocol equipped with the assay kit, which was purchased from Promega (Madison, WI, USA).

Cytokines assay

The primary keratinocytes obtained were cultured in 96-well microtiter plates (1 × 10 3 cells/well), and incubated for 24 h in the presence of TNF-α (1μg/ml). Then supernatants of culture medium was collected and IL-6, IL-8, EGF, HGF, TGF-α, epiregulin, and amphiregulin were measured by enzyme-linked immunosorbent assay (ELISA) kits (BioSource). These assays detected only human cytokines and the minimal detectable concentrations were 2.4pg/ml for TNF-α, 1.5 pg/ml for IL-6, 1.5pg/ml for IL-8, 3.0pg/ml for EGF, 50pg/ml for HGF, 0.2pg/ml for epiregulin, and 0.25pg/ml for amphiregulin, respectively.

Statistics

Statistical significance of the data obtained was evaluated by Student's t -test using unpaired analyses.

Materials

Dulbecco's modified Eagle's medium and KGM medium were purchased from GIBCO (Grand Island, NY, USA). Penicillin and streptomycin were obtained from M.A. Bioproducts (Walkersville, MD, USA). All other chemicals were purchased from Nakarai Chemicals Ltd. (Kyoto, Japan).


   Results Top


Cell growth of primary keratinocytes in the presence of various cytokines

The effect of various cytokines and growth factors on the growth of primary keratinocytes of normal (NK), psoriatic involved (PIK), and uninvolved skin (PUK) was determined. IL-6, IL-8, EGF, HGF, TGF-α, epiregulin, and amphiregulin but not TNF-α significantly increased cell proliferation of these keratinocytes [Figure 1]. The increased cell proliferation was most potent in EGF-treated keratinocytes. The increased cell proliferation by these cytokines and growth factors were not different among NK, PIK, and PUK [Figure 1].

Cytokines and growth factors secretion of TNF-a-treated keratinocytes

The effect of TNF-α on cytokines and growth factors secretion of keratinocytes was determined. TNF-α significantly induced the secretion of IL-6, IL-8, EGF, HGF, TGF-α, epiregulin, and amphiregulin up to 2-8-fold after 24 h [Figure 2]. The effect was observed up to 48 h (data not shown). The increased levels of cytokines and growth factors by TNF-α were not different among NK, PIK, and PUK [Figure 2].


   Discussion Top


Psoriatic hyperproliferative epidermis has been characterized by increased keratinocyte proliferation accompanied by characteristic histopathology. [5],[6] The induction of increased keratinocytes is induced by several growth factors and cytokines, such as, EGF, amphiregulin, epiregulin, IL-6, and IL-8. In addition, histochemical studies have indicated the increased local levels of various cytokines. [1],[2],[3] In the present study, we showed that EGF, amphiregulin, epiregulin, IL-6, and IL-8 increased keratinocytes proliferation confirming the previous studies.

Although T cells show an essential role in the induction and maintenance of psoriasis, it is still not clear that whether the keratinocytes derived from psoriasis show distinct behavior compared to normal keratinocytes. Oyama et al . showed the difference of EGF receptor expression induced by IL-6 between normal and psoriatic lesional keratinocytes. [7] They suggested different EGF receptor regulatory system regarding the pathogenesis of psoriasis. Several studies revealed the lack of effect of TNF-α on proliferation and differentiation in healthy and psoriatic keratinocytes. Also studies have reported that psoriatic keratinocytes are equally responsive to the stimulatory effects of TGF-α and IL-8, but are less susceptible to IL-6. [8],[9] In the present study, increased keratinocytes proliferation induced by EGF, amphiregulin, epiregulin, IL-6, and IL-8 were not significantly different among the normal, psoriatic lesional, and nonlesional keratinocytes, thereby conforming the previous studies. Furthermore, TNF-α-induced secretion of cytokines was not different among these keratinocytes. These results suggest that the keratinocytes from psoriasis do not reveal distinct behavior regarding cell proliferation and TNF-α dependent cytokine secretion at least for the tested cytokines and that for T cells and dendritic cells but not keratinocytes are more essential for the pathogenesis of psoriasis. Finally, the recent findings suggest the essential role of Th17 cells on the pathogenesis of psoriasis. [10],[11] The marked increase in IL-6 secretion by keratinocytes is quite interesting, because this cytokine is required for the Th17 cell induction. [10]


   Acknowledgments Top


We thank Mss Yasuyo Nishinome, Mrs. Keiko Nishikura, and Miss Miharu Watanuki for providing technical support for our study.

 
   References Top

1.Nickoloff BJ. The cytokine network in psoriasis. Arch Dermatol 1991;127:871-84.  Back to cited text no. 1      
2.Krueger G, Ellis CN. Psoriasis; recent advance in understanding its pathogenesis and treatment. J Am Acad Dermatol 2005;53:S95-S100.  Back to cited text no. 2      
3.Lowes MA, Bowcock AM, Krueger JG. Pathogenesis and therapy of psoriasis. Nature 2007;445:866-73.   Back to cited text no. 3      
4.Nickoloff BJ, Mitra RS, Elder JT, Fisher GJ, Voorhees JJ. Decreased growth inhibition by recombinant gamma interferon is associated with increased transforming growth factor-production in keratinocytes cultured from psoriatic lesion. Br J Dermatol 1989;121:161-74.  Back to cited text no. 4      
5.Iizuka H, Ishida-Yamamoto A, Honda H. Epidermal remodeling in psoriasis. Br J Dermatol 1996;135:433-8.  Back to cited text no. 5      
6.Iizuka H, Honda H, Ishida-Yamamoto A. Epidermal remodeling in psoriasis (II): A quantitative analysis of the epidermal architecture. J Invest Dermatol 1997;109:806-10.  Back to cited text no. 6      
7.Oyama N, Sekimata M, Nihei Y, Iwatsuki K, Homma Y, Kaneko F. Different growth properties in response to epidermal growth factor and interleukin-6 of primary keratinocytes derived from normal and psoriatic lesional skin. J Dermatol Sci 1998;16:120-8.  Back to cited text no. 7      
8.Fransson J. Tumour necrosis factor-alpha does not influence proliferation and differentiation of healthy and psoriatic keratinocytes in a skin-equivalent model. Acta Derm Venereol 2000;80:416-20.  Back to cited text no. 8      
9.Olaniran AK, Baker BS, Garioch JJ, Powles AV, Fry L. A comparison of the stimulatory effects of cytokines on normal and psoriatic keratinocytes in vitro. Arch Dermatol Res 1995;287:231-6.  Back to cited text no. 9      
10.Nickoloff BJ. Cracking the cytokine code in psoriasis. Nat Med 2007;13:242-4.  Back to cited text no. 10      
11.Zaba LC, Cardinale I, Gilleaudeau P, Sullivan-Whalen M, Suárez-Fariñas M, Fuentes-Duculan J, et al . Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses. J Exp Med 2007;204:3183-94.  Back to cited text no. 11      


    Figures

  [Figure 1], [Figure 2]

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    Abstract
    Introduction
    Materials and Me...
    Results
    Discussion
    Acknowledgments
    References
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