Differential Upregulation and Functional Activity of S1PR1 in Human Peripheral Blood Basophils of Atopic Patients

Abstract

Basophils are key effector cells in atopic diseases, and the signaling sphingolipid Sphigosine-1-phosphate (S1P) is emerging as an important mediator in these conditions. The possible interaction of S1P and basophils and the resulting biological effects have not yet been studied. We hypothesize that S1P influences the function of basophils in atopy and aim to elucidate the modes of interaction. S1P receptor (S1PR) expression in human peripheral blood basophils from atopic and non-atopic patients was assessed through qRT-PCR and flow cytometry analysis. Functional effects of S1P were assessed through a basophil activation test (BAT), calcium flux, apoptosis, and chemotaxis assays. Immunofluorescence staining was performed to visualize intracellular S1P. Human basophils express S1PR1, S1PR2, S1PR3, and S1PR4 on the mRNA level. 0.1 µM S1P have anti-apoptotic, while 10 µM exhibits apoptotic effects on basophils. Basophils from atopic patients show less chemotactic activity in response to S1P than those from healthy donors. Protein expression of S1PR1 is downregulated in atopic patients, and basophils in lesional AD skin possess intracellular S1P. These findings suggest that the interaction of S1P and basophils might be an important factor in the pathophysiology of atopy.

TRPV1 Channel in Human Eosinophils: Functional Expression and Inflammatory Modulation

“…Experiments to analyze changes in calcium flux were performed according to the same procedure as described recently [67]. Purified eosinophils were labeled with 3 µM Fluo-4 (Molecular Probes, Eugine, OR, USA) and stimulated with 0.1, 1, 10, or 100 µM capsaicin (Merck, Darmstadt, Germany) during the measurement.…”

Basophils in pruritic skin diseases

“…Due to the inhibiting effect of the lipid mediator on chemotaxis, S1P is proposed to have an anti-inflammatory effect on basophils (31).…”

“…SP has also been shown to chemoattract basophils, resulting in the infiltration of basophils into the skin of healthy individuals (49). Recently, it was shown that in healthy donors, basophils migrate towards S1P which was observed in an in vitro study, while in AD patients a chemorepulsive effect was detected (31). It has however, so far not been described if basophils, that migrated into the skin, return to the blood or travel to draining lymph nodes (45,50).…”

Density Gradient Centrifugation‐Independent Purification of Human Basophils

“…(2023) and Gray et al. (2022). Furthermore, we describe a flow cytometry staining protocol to assess basophil yield, purity, and viability (Support Protocol 1), as well as a staining protocol to visually inspect the purified basophils (Support Protocol 2).…”

“…The manufacturers' protocol was modified to use a lower amount of antibody cocktail and magnetic beads, therefore reducing the cost, but still ensuring cell purities to be consistently >96%. We have used this method for publishing our research on basophils in Limberg et al (2023) and Gray et al (2022). Furthermore, we describe a flow cytometry staining protocol to assess basophil yield, purity, and viability (Support Protocol 1), as well as a staining protocol to visually inspect the purified basophils (Support Protocol 2).…”