Yoshihiko Soga describes for SIIC his article published in Asian Pacific Journal of Allergy and Immunology 31(4):271-276, December 2013

Okayama, Japón (special for SIIC)
Eosinophil cationic protein (ECP) is one of the proteins released on eosinophil degranulation, and was detected in allergic inflammatory lesions. Thus ECP was reported previously to be involved in allergic inflammation with cytotoxic activity. This cytotoxicity is mainly explained as an antiparasitic and bactericidal activity in infectious diseases. However, ECP has also been reported to be cytotoxic toward host cells, e.g., the tracheal epithelium. On the other hand, recent studies showed that ECP did not induce cell death but inhibited the growth of cancer-derived cells. Our previous study indicated that human ECP enhanced differentiation of rat neonatal cardiomyocytes and stress fiber formation in Balb/c 3T3 mouse fibroblasts, while the effects of human ECP on human fibroblasts are unknown. Because, despite their usefulness in determination of eosinophil physiology in vivo, murine models differ from human in several major respects. For example, airway mucosal eosinophils in allergen-challenged mice lack signs of degranulation. Thus, the role of human ECP could be different from that in rodents. Therefore, it is important to evaluate the effects of human ECP on human fibroblasts.

The present study was performed to determine the effects of human ECP on cytokine expression on normal human dermal fibroblasts (NHDF) by assay for cell growth. Furthermore, cytokine expression of NHDF stimulated by ECP, which could influence cell growth was evaluated by protein array.

Recombinant human ECP (rhECP) in mature form without the secretion signal peptide was expressed in bacteria and prepared as described previously. Commercially available adult NHDF were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 10 µg/ml gentamycin at 37°; under a humidified 5% CO₂ atmosphere. All in vitro studies using NHDF were performed between passages 5 and 9.

NHDF were plated into 8-well chamber slides with DMEM containing 10% FBS and 10 µg/ml gentamycin at 5.0×10³ cells/well. After 24 h, the medium was changed to DMEM containing 0.5% FBS without gentamycin. After a further 24 h, cells were stimulated with rhECP (0 - 10 µg/ml) for 24 h. The cells were stained with hematoxylin-eosin, and microscopic observation of NHDF with or without rhECP (0 - 10 µg/ml) stimulation was performed. Furthermore, the number of NHDF cells was counted at a magnification of × 100. Five separate fields from a single well were counted, and five separate wells were used for each rhECP concentration group. Averages and standard deviations were calculated from three independent experiments. The cell growth of NHDF was evaluated using microscopic observation and cell counts of NHDF stimulated with rhECP.

NHDF were plated into 6-well multiple well plates with DMEM containing 10% FBS and 10 µg/ml gentamycin at 1.0×10⁵ cells/well. After 24 h, the medium was changed to DMEM containing 0.5% FBS without gentamycin. After a further 24 h, cells were stimulated with or without 100 ng/ml rhECP for 24 h. Culture supernatants were obtained, and cytokine measurements were performed by human cytokine antibody array C series 1000), consisting of 120 different cytokine and chemokine antibodies spotted in duplicate onto a membrane according to the manufacturer’s instructions. The array was exposed to film and the intensity of signals was detected using ImageJ software. The relative intensity levels of the cytokines were normalized with reference to the amounts present on the positive control in each membrane based on the average of cytokine spot intensity levels divided by the average of the positive control spot intensity levels and are shown as percentages. The sensitivities of the cytokines examined are provided on the manufacturer’s website. Average and standard deviations were calculated from three independent experiments.

ECP was not cytotoxic toward NHDF, but rather enhanced the growth of these cells. A dose-dependent increase in cell count was observed. The peak rhECP concentration that enhanced the cell counts by 1.56-fold was 100 ng/ml, which was significantly different from cultures without ECP stimulation (ANOVA/ Scheffe’s test, p < 0.05). Array analyses indicated that ciliary neurotrophic factor (CNTF), neutrophilactivating peptide (NAP)-2, and neurotrophin (NT)-3 were significantly upregulated in NHDF stimulated with 100 ng/ml ECP compared to those without stimulation (Welch’s test, p < 0.05).

The results of the present study indicated that ECP is not cytotoxic but enhances the growth of NHDF, which was consistent with our previous observation in Balb/c 3T3 fibroblasts. All three significantly upregulated molecules-CNTF, NAP-2, and NT-could explain the involvement of ECP in allergic inflammation and the promotion of growth of NHDF. Indeed, while not particularly high (1.56-fold), the enhancement of cell count by ECP stimulation was observed. These findings regarding ECP activity suggest that ECP functions in fibrosis with mild promotion of allergic inflammation. Other molecules that were upregulated below the level of significance on array analysis may also contribute to this phenomenon in a cooperative manner. Our results also indicated that the levels of production of major proinflammatory cytokines, such as IL-beta, IL-6, TNF-alpha, etc., were not significantly increased by ECP stimulation. Taken together, these observations indicated that ECP is not cytotoxic and does cause a strong inflammatory reaction in NHDF.

In conclusion, ECP is not cytotoxic but enhances the growth of NHDF. The results of array analyses indicated that CNTF, NAP-2, and NT-3 were suggested to be involved in enhancing the growth of NHDF. These findings will contribute to determination of the role of ECP in allergic inflammation.