Instructions for use Title Increase in macrophage migration inhibitory factor levels in lacrimal fluid of patients with severe atopicdermatitis

Background and aims of the study: Atopic dermatitis is a chronic inflammatory skin disorder that frequently involves some ophthalmic features. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is associated with the generation of cell-mediated immune responses. Although serum MIF levels may be elevated in severe atopic dermatitis, the quantity of MIF in regional ocular fluid remains unknown. We measured MIF levels in tears (lacrimal fluid) of patients with atopic dermatitis. Patients and Methods: Tear samples were collected from 16 patients with atopic dermatitis, 10 patients with allergic conjunctivitis, and 15 healthy control subjects. The clinical severity of atopic dermatitis was evaluated according to the Scoring Atopic Dermatitis (SCORAD) index. The index was calculated to sum up following scores: extent criteria, intensity criteria, and subjective symptoms. Macrophage migration inhibitory factor levels were determined by a human MIF enzyme-linked immunosorbent assay. All comparisons were made two-tailed, and p-values < 0.01 were considered as statistically significant. Results: The mean MIF concentration in lacrimal fluid collected from healthy control subjects was 0.69 ± 0.2 ng/ml. The mean tear MIF levels were 17.87 ± 6.3 ng/ml in moderate-severe atopic dermatitis (SCORAD ≥ 15, p=0.002), 0.93 ± 0.08 ng/ml in mild atopic dermatitis (SCORAD < 15), and 2.76 ± 0.86 ng/ml in allergic conjunctivitis (p=0.008). Conclusions: A proinflammatory cytokine MIF level was elevated in tears as well as serum in cases of severe atopic dermatitis. These results suggest that MIF may play an important role in the induction or enhancement of ophthalmic features caused by severe atopic dermatitis. Introduction Macrophage migration inhibitory factor (MIF) was first discovered in the late 1960s; it is therefore believed to be the first lymphokine [6]. Since MIF was discovered as a part of the phenomenon, not a material, to inhibit migration of macrophages in the pre-molecular biology era, many scientists doubted its importance in the immune response. Investigations in the 1990s aimed at identifying novel systemic mediators that could regulate host inflammatory responses led to the identification of murine MIF as a product secreted by the anterior pituitary gland [2]. Upon stimulation, T cells release MIF, and MIF activity was first described as a product of cognate T cell supernatants [15]. Macrophages are also now identified as an important source of MIF and are known to express MIF both constitutively and upon stimulation [15]. Macrophage migration inhibitory factor is considered to act by both paracrine and autocrine stimulatory pathways to augment the activation of these cells [15]. As reported previously, MIF is essential for T cell activation and possibly contributes to maintaining Th1/Th2 imbalance [1]. Increased MIF expression has been reported in lesions from many immune/ inflammatory diseases, including psoriasis, glomerulonephritis, transplant rejection, neuro-Behçet’s disease, asthma, adult respiratory distress syndrome, and inflammatory eye diseases [3, 4, 7, 11, 12, 13, 16, 17, 20, 24]. Atopic dermatitis (AD) frequently involves some ophthalmic features: blepharitis, chronic keratoconjunctivitis, keratoconus, early-onset cataract, and rarely, retinal detachment [9]. AD is a chronic inflammatory skin disorder and many reports have documented its pathogenesis in relation to genetic and immunological abnormalities as well as environmental factors [10]. Although abnormal populations of Th1 and Th2 subsets of helper T cells (Th1/Th2 imbalance) had been identified as a cause of the pathogenesis of AD [8, 13, 25], a decrease in delayed-type hypersensitivity (DTH) is considered to involve more than Th1/Th2 imbalance in AD [5]. MIF is essential for T cell activation and possibly contributes to maintaining Th1/Th2 imbalance as described above [1]. Also, a prominent increase in systemic MIF levels was detected in patients with severe AD, and the levels decreased when the clinical symptoms improved following treatment with corticosteroid ointment [18, 19]. We hypothesized that a high concentration of MIF could exist in the regional fluid of the eye as well as in serum in cases of severe atopic dermatitis. Since Scoring Atopic Dermatitis (SCORAD) index, which is determined on the basis of several criteria concerning lesion spread and intensity as well as subjective signs, is commonly used to evaluate AD [21], AD patients were classified into two groups as moderate-severe and mild AD according to SCORAD index in the present study. We measured the MIF levels in tears (lacrimal fluid) of patients with AD and compared them to those of patients with allergic conjunctivitis (AC) and healthy people. Materials and Methods Patients We studied 16 patients with AD and 9 subjects with AC who visited the Departments of Dermatology and Ophthalmology, Hokkaido University Hospital, Sapporo, Japan. Atopic dermatitis is a common inflammatory disorder characterized by a chronic and relapsing course. In order to evaluate the severity of the disease as objectively as possible, the European Task Force on Atopic Dermatitis had developed a method allowing consistent assessment by means of a severity index, called Scoring Atopic Dermatitis (SCORAD) index [21]. The index should be calculated to sum up following socres: (1) extent criteria (involved surface area), (2) intensity criteria (erythema, edema/ papulation, oozing/ crusting, excoriation, and lichenification), and (3) subjective symptoms (pruritus and insomnia) [21]. We classified cases of AD as “moderate-severe” (SCORAD ≥ 15) or “mild” (SCORAD < 15) according to the SCOARD index in this study. Each patient with moderate-severe AD had atopic manifestations on the facial skin. Allergic conjunctivitis (AC) was diagnosed by slit lamp examination according to the guidelines of diagnosis and treatment of conjunctivitis, reported elsewhere [23]. Though we collected tear samples out of pollen season (December, January, and February), five of ten AC patients had sensitivity to grass or birch pollen by interview. Most of the AC patients were considered as chronic phase of AC, and their conjunctival signs and symptoms were mild. Tear samples were collected from 9 patients with severe AD (mean age, 26.1 years; age range, 18-37 years), 7 patients with mild AD (mean age, 29.0 years; age range, 16-44 years), 10 patients with allergic conjunctivitis (AC) (mean age, 32.6 years; age range, 22-44 years), and 15 healthy volunteers (mean age, 34.6 years; age range, 23-45 years). All subjects were Japanese, and healthy volunteers with no history of AD were recruited from our colleagues as controls. Dermatologists and ophthalmologists also verified no manifestations of AD and AC in controls, when their tear/ serum samples were collected. Informed consent was obtained from every patient and control subject. Collection of tears and sera All of the experiments in this study followed the tenets of the Declaration of Helsinki. After informed consent was obtained, tear samples were collected from all subjects. To obtain unstimulated basal lacrimal fluid, the tear samples (10 μl) were collected with microcapillary tubes for microhematocrit (75 mm length, Funakoshi Ltd., Tokyo) at the lateral canthus of patients in the supine position without any anesthesia. After obtaining informed consent, serum samples were collected from two of severe AD patients whose tear MIF levels were quite high (> 27.2 ng/ml), exceeded 1-standard deviation (SD) from the group’s median value. Also, two subjects were chosen randomly from each group of the patients with mild AD and healthy controls to measure their serum MIF levels. Tear samples were centrifuged immediately at 4°C to remove cells and transferred into new tubes. Tear and serum samples were stored at -80°C until further examination. Measurement of MIF Macrophage migration inhibitory factor levels were determined by a human MIF enzyme-linked immunosorbent assay (ELISA) (CosmoBio, Tokyo, Japan) as described previously [18]. It contains all reagents necessary for performing the assay. Statistical analysis was performed using the Mann-Whitney U test.