Epiderm, a human three-dimensional epidermal model, was exposed to 50 mJ per cm 2 UVB irradiation. The culture supernatants were obtained before and 1, 6, 12, 24, and 48 h after UVB irradiation and their MIF contents were measured by ELISA as described in Materials and Methods . MIF contents with ( d ) and without ( s ) UVB irradiation. Results are expressed as mean Ϯ SD of three different experiments. 

Contexts in source publication

Context 1

... culture media of lower chambers were obtained before and after UVB irradiation at 1, 6, 12, 24, and 48 h. The results showed that MIF was readily released into the medium from 1 h after the irradiation, and formed biphasic maximal peaks at 6 and 48 h after the exposure (Fig 3). ...

Context 2

... skin is an important physical barrier that protects the body from the damage caused by the outside environment, including trauma, bacterial infection, and ultraviolet (UV) irradiation. As for the biologic mechanism, the skin is known to secrete a number of cytokines that act as immunologic and inflammatory mediators for self-defense. In particular, keratinocytes, which constitute the majority of the cell mass of the epidermis, are a major source for a wide array of these cytokines ( Sauder et al , 1990). Indeed, there is emerging evidence that keratinocytes participate in cutaneous inflammatory reactions and immune responses by producing a variety of cytokines. UV irradiation may trigger cutaneous inflammatory responses by stimulating epidermal keratinocytes to produce biologically potent cytokines such as interleukin (IL)-1 (Kupper et al , 1987; Ansel et al , 1988), IL-6 (Kirnbauer et al , 1991), and tumour necrosis factor (TNF)- α (K ̈ ck et al , 1990). These cytokines are involved not only in the mediation of local inflammatory reactions within the epidermis, but also exert systemic effects through entrance into the circulation. In a previous study, we have shown by reverse transcriptase- polymerase chain reaction (RT-PCR) and immunoblot analysis that human epidermal keratinocytes express macrophage migration inhibitory factor (MIF). Immunohistochemical study revealed that MIF exists in human epidermis, especially in the basal layer (Shimizu et al , 1996). This finding suggested the possibility that MIF regulates epidermal immunity, inflammation, and cellular differentiation. Human MIF cDNA was first cloned from activated T cells, demonstrating that this protein consists of 114 amino acid residues (Weiser et al , 1989). Following this finding various novel properties of MIF have been elucidated. It was reported that MIF is mainly expressed in T lymphocytes and macrophages; however, recent studies have revealed that this protein is ubiquitously expressed in various cells (Lanahan et al , 1992; Wistow et al , 1993; Onodera et al , 1995; Imamura et al , 1996; Suzuki et al , 1996). To date, MIF is known to respond to stimuli such as wounds and infection and is considered to contribute to regulation of inflammatory and immunologic responses to such tissue damage (Bernhagen et al , 1993; Calandra et al , 1994). Recently, it was reported that endotoxin administration induced expression of MIF protein and mRNA in various tissues of the rat (Bacher et al , 1997). As for the environmental effects on the skin, the most relevant physical injury to human skin is UVB radiation. MIF produced by keratinocytes may be profoundly involved in cutaneous inflammatory responses; however, the precise pathophysiologic function of MIF in the skin still remains unclear. UVB radiation constitutes one of the most important physiologic modulators of inflammatory and immunologic processes in human skin. We, therefore, examined the production of MIF induced in keratinocytes by UVB in vivo and in vitro to evaluate its pathophysiologic role. RESULTS In vitro UVB exposure At 24 and 48 h after UVB irradiation, a significant decrease of viability was observed only after more than 100 mJ per cm 2 UVB irradiation (data not shown). To examine the release of MIF caused by UVB exposure, we irradiated the cultured keratinocytes with various amounts of UVB (0–125 mJ per cm 2 ). A significant increase of MIF secretion in the supernatants of cultured keratinocytes was detected at 48 h after UVB radiation in a concentration-dependent manner up to 100 mJ per cm 2 ( Fig 1 A ). Similarly, a significant increase of TNF- α release was demonstrated after UVB radiation until 75 mJ per cm 2 ( Fig 1 B ). Extracellular secretion of MIF was observed at 1 h after UVB irradiation with 50 mJ per cm 2 , and increased in a time-dependent manner up to 48 h ( Fig 2 A ). On the other hand, the release of TNF- α after UVB irradiation reached its maximum at 24 h ( Fig 2 B ). To examine in more detail whether UVB exposure could induce MIF release from the multilayered epidermis, EpiDerm, a three-dimensional epidermis with highly differentiated charac- teristics, was exposed to 50 mJ per cm 2 UVB irradiation through the air–medium interface. The culture media of lower chambers were obtained before and after UVB irradiation at 1, 6, 12, 24, and 48 h. The results showed that MIF was readily released into the medium from 1 h after the irradiation, and formed biphasic maximal peaks at 6 and 48 h after the exposure ( Fig 3 ). MIF content in human epidermal sheet culture supernatants was assessed before and after UVB irradiation. Significant amounts of MIF were detected 12–24 h after 50 mJ per cm 2 UVB irradiation, 44.2 Ϯ 6.0 and 108.5 Ϯ 12.5 ng per ml, respectively ( Fig 4 ). These results were confirmed by immunohistochemical analysis at 12 h after UVB irradiation. This revealed extensive positive staining of all the layers in the epidermis with the anti-MIF antibody ( Fig 5 b ). On the other hand, predominant staining was observed mostly in the basal layer for sham-irradiated skin ( Fig 5 a ) as described previously (Shimizu et al , 1996). No specific positive staining was observed when the tissue was reacted with nonimmune rabbit IgG (data not shown). To examine whether the induction of MIF by human epidermis is regulated at the transcriptional level, the expression of MIF mRNA in cultured keratinocytes was examined by northern blot analysis after 50 mJ per cm 2 UVB exposure. Keratinocytes constitutively expressed MIF mRNA without exposure, and the expression level was upregulated during 1–6 h by 1.4–2.1-fold in comparison with the basal level (0 h) after the UVB exposure and slightly decreased at 12 h ( μ 0.8-fold) ( Fig 6 ). Following this, the expression level was restored to the basal level during 24–48 h. In vivo UVB exposure When serum MIF content was investi- gated in human volunteers before and after UVB exposure (four MED), minimal levels of MIF content could be detected before irradiation (10.8 Ϯ 1.5 ng per ml). After UVB exposure, significantly elevated serum MIF was detected at 48 h (58.1 Ϯ 20.6 ng per ml) ( Fig 7 ). MIF levels were biphasic after the UVB exposure, even though there were some individual differences ( Fig 8 a – e ). The first peak was observed between 1 and 12 h after irradiation and ranged from 15.1 to 82.8 ng per ml (early response). The second peak of MIF content was observed at 12–48 h after UV irradiation and ranged from 24.1 and 132.6 ng per ml (late response) when clinical signs of sunburn were clearly observed. DISCUSSION It has been reported that various cytokines play important parts in the homeostasis of normal skin and in its pathologic states (Barker et al , 1991). Release of cytokines by keratinocytes can be induced by a number of environmental factors, including UV light, trauma, and bacterial toxins. It is well recognized that excessive exposure to sunlight results in a number of deleterious consequences such as erythema and inflammation of the skin. Moreover, UV radiation has a direct carcinogenic effect, which indicates that exposure to sunlight can result in profound immunologic alterations (Scotto et al , 1974). There is increasing evidence that keratinocytes participate in cutaneous immunologic and inflammatory reactions through production of a variety of cytokines. To date, keratinocytes have been shown to secrete several UVB-induced cytokines such as IL-1, IL-3, IL-6, IL-10, IL-15, and TNF- α (Kupper et al , 1987; Ansel et al , 1988; Oxholm et al , 1988; K ̈ ck et al , 1990; Gallo et al , 1991; Kirnbauer et al , 1991; Enk et al , 1993, 1995; Mohamadzadeh et al , 1995). For example, TNF- α is an immunologically potent cytokine regulating immunologic and inflammatory responses of the skin (Oxholm et al , 1988; K ̈ ck et al , 1990), and TNF- α and IL-10 released from keratinocytes subsequent to UVB exposure may play a part in the induction of immunosuppression and tolerance (Yoshikawa and Streilein, 1990; Enk et al , 1993; Shimizu and Streilein, 1994). This study describes enhancement of MIF production in the skin by UVB irradiation in vivo and in vitro . It is of interest that two peaks of serum MIF content were observed after UVB exposure in vivo . The first peak was observed at 1–12 h (early response) after the exposure and the second peak at 12–48 h (late response). In vitro , we carried out northern blot analysis to examine the effect of UVB on the expression of MIF mRNA using cultured keratinocytes, and observed upregulation of the MIF mRNA level during 1–6 h after UVB exposure, but a slight decrease at 12 h. Thereafter, the expression level recovered to the basal level during 24–48 h. It was reported that positive MIF immunostaining of cultured pituitary cells disappeared after 16 h of incubation with lipopolysaccharide (LPS) and, in contrast, MIF mRNA expression was increased (Bernhagen et al , 1993). Similarly, intracellular MIF of macrophages disappeared concomitant with the increase of MIF in the culture medium in response to LPS (Calandra et al , 1994). Consistent with these findings, we found an increase of MIF content in the culture medium after UVB exposure. Based on these facts, it is speculated that the major part of the increased serum MIF content in the early response after UVB exposure in vivo may correspond to the release of MIF protein from keratinocytes. On the other hand, the source of increased serum content MIF in the late response has ...