Innovative approaches to study basophil function in inflammation

Abstract: Basophils are circulating granulocytes. They are very rare and represent less than 1% of peripheral blood leukocytes. Basophils connect the innate and adaptive immune responses by the secretion of a variety of immune-mediators involved in the pathogenesis of many inflammatory diseases mainly allergic reactions and autoimmune diseases. For many years it was difficult to study basophil function due to their rareness in peripheral blood which resulted in scant yields and purity when isolated. This thesis focuses on studies of the basophil function in two inflammation-driven diseases: chronic kidney disease (CKD) and allergy. The investigations have been done using newly developed microfluidic-based lab-on-chip technology and conventional immunological methods. In paper I, we investigated the impact of blood-membrane interaction on circulating basophils and neutrophils in hemodialysis patients (stage 5D), using high-flux and low-flux dialyzers. Passage through the low-flux dialyzer, as opposed to high-flux, induced a significant upregulation of CD63 on formyl-methyinoyl-leucyl-phenylalanine (fMLP) and anti-FcεRI antibody stimulated basophils. Furthermore, (fMLP) stimulated basophils significantly upregulated CD63, in patients compared to healthy controls. There were no significant differences in the expression of neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88), when comparing the two dialyzers, or when compared to healthy controls. In paper II, we analyzed the expression of activator markers on basophils related to two crucial functions (transmigration and degranulation) in CKD (stage 5D). The CD300a expression was significantly higher in patients following activation by fMLP and anti-FcɛRI-ab and the expression of the active epitope of CD11b was significantly higher in patients after lipopolysaccharide (LPS) activation. The CD62L expression was significantly downregulated in anti-FcɛRI activated basophils from healthy controls. In paper III, we developed a novel microfluidic immuno-affinity based basophil activation test (miBAT) assay. The microfluidic device is capable of isolating basophils directly from whole blood and we analyzed the regulation of CD203c and CD63 in anti-FcεRI activated basophils in healthy and allergic individuals. The microfluidic chip was able to capture basophils from whole blood with an efficiency of 65% and the CD63 expression detected via fluorescent microscope was significantly higher in activated basophils compared to non-activated basophils (negative control), as well as in allergic patients compared to healthy controls in microfluidic chip. The result was comparable to flow cytometry data. In paper IV we validated that the miBAT platform can be used for allergy diagnosis. CD63 expression on basophils activated with allergens was detected in microfluidic chip and flow cytometry. The activation was significantly higher compared to non-activated basophils in allergic patients. Basophils from non-allergic individuals did not respond to allergen activation. The microfluidic chip analysis was comparable with flow cytometry data. In conclusion, this thesis presents new insights on the role of basophils in the inflammatory responses, mainly related to innate immune responses in CKD patients. Moreover, we introduced a novel microfluidics based method (miBAT) to quantify basophil activation in allergic patients. The method has great potential to be used as a point of care for allergy diagnosis.