The expression patterns of IL-1ß and IL-10 and their relation to CYP epoxygenases in normal human placenta.

INTRODUCTION: The success of pregnancy depends on the regulation of immunological processes in the placenta. Important mediators of an immune response include pro- and anti-inflammatory interleukins which may be regulated by CYP epoxygenases and their metabolites. The relation between interleukins and CYP epoxygenases expression in human placenta has not yet been studied vastly. MATERIAL AND METHODS: We investigated the expression patterns of IL-1ß and IL-10 in embryonic (n=8), early foetal (n=16) and term (n=7) human placenta tissue by an immunohistochemical method and evaluated the results by Kruskal-Wallis test. The obtained data was correlated using Spearman's correlation coefficient to our previously published data of CYP epoxygenases expression in the same samples. To confirm that Hofbauer cells express IL-10 and IL-1ß as well as CYP2C8 and IL-10 together, and thus there is a relation between proteins of interests, we used multiplex immunofluorescent staining. RESULTS: The expression of IL-1ß decreased with gestational age in cytotrophoblast, syncytiotrophoblast, as well as in Hofbauer cells whilst IL-10 decreased in cytotrophoblast, remained at the same levels in syncytiotrophoblast and increased in Hofbauer cells. In trophoblast cells, we found a statistically significant positive correlation between the expression of CYP2J2 and CYP2C9 with IL-1ß, whereas there was no relation between IL-10 and any of the tested CYP epoxygenases. In Hofbauer cells, we found a significant positive correlation between CYP2C8 and IL-10 and a significant negative correlation between CYP2C8 and IL-1ß. CONCLUSION: Our results showed that the exact role and relation of interleukins and CYP epoxygenases and their metabolites is dependent on their respective cellular context. Because of IL-10, IL-1ß, as well as HBCs play a role in various pathological conditions, further investigation of the exact role of CYP epoxygenase, interleukins and their relations is needed.

Beneficial Effect of Heat-induced Antigen Retrieval in Immunocytochemical Detection of Intracellular Antigens in Alcohol-fixed Cell Samples.

Immunohistochemistry and immunocytochemistry (ICC) play an irreplaceable role in research and diagnostics. It is well known that antigen retrieval (AR) can, as a technique, have beneficial outcomes on immunohistochemistry results when using formalin-fixed, paraffin-embedded tissue samples. The main purpose of AR is to break protein crosslinks which are formed during formalin fixation. Although AR was originally designed for formalin-fixed, paraffin-embedded samples, the usefulness of AR in ICC has been described in previous studies. Cytologic samples are often fixed in alcohol-based fixatives which does not lead to the formation of crosslinks. Therefore, alcohol-fixed samples can be successfully immunostained without AR. We investigated the effect of heat-induced antigen retrieval (HIAR) on alcohol-fixed HEK293 cell line samples and patient cytologic samples from thyroid gland obtained by fine needle aspiration technique. We compared indirect 2-step ICC staining results performed according to the protocol with or without HIAR in citrate buffer pH 6 for several antibodies. Utilizing HIAR against intracellular antigens has beneficial effects. Therefore, more diluted antibodies can be used for satisfactory results. However, surface antigens were probably damaged by HIAR treatment. We demonstrated evident changes in cell surface topography after HIAR treatment by atomic force microscopy. Staining specificity of patient samples improves and background staining is reduced, allowing higher dilutions of primary antibody. Improving staining specificity is necessary for accurate diagnostics. Although we have shown the beneficial effect of HIAR for immunostaining intracellular antigens, proper staining protocol should be tested on appropriate controls for individual antibodies.