Markers Involved in Innate Immunity and Neutrophil Activation are Elevated during Acute Human Anaphylaxis: Validation of a Microarray Study

Abstract

Background: We have previously identified the upregulation of the innate immune response, neutrophil activation, and apoptosis during anaphylaxis using a microarray approach. This study aimed to validate the differential gene expression and investigate protein concentrations of "hub genes" and upstream regulators during anaphylaxis.

Methods: Samples were collected from patients with anaphylaxis on their arrival at the emergency department, and after 1 and 3 h. mRNA levels of 11 genes (interleukin-6 [IL-6], IL-10, oncostatin M [OSM], S100A8, S100A9, matrix metalloproteinase 9 [MMP9], FASL, toll-like receptor 4 [TLR4], MYD88, triggering receptor expressed on myeloid cells 1 [TREM1], and cluster of differentiation 64 [CD64]) were measured in peripheral blood leucocytes using qPCR. Serum protein concentrations were measured by ELISA or cytometric bead array for 6 of these candidates.

Results: Of 69 anaphylaxis patients enrolled, 36 (52%) had severe reactions, and 38 (55%) were female. Increases in both mRNA and protein of IL-10, S100A9, MMP9, and TREM1 were observed. OSM, S100A8, TLR4, and CD64 were upregulated and IL-6 protein concentrations were increased during anaphylaxis. Both FASL and soluble Fas ligand decreased during anaphylaxis.

Conclusion: These results provide evidence for the involvement of innate immune pathways and myeloid cells during human anaphylaxis, validating previous microarray findings. Elevated S100A8, S100A9, TLR4, and TREM1 expression, and increased S100A9 and soluble TREM1 protein concentrations strongly suggest that neutrophils are activated during acute anaphylaxis.

Keywords: Allergy; Anaphylaxis; Myeloid cells; Neutrophils.

Fig. 1

Gene expression during anaphylaxis. Quantitative PCR was performed on mRNA extracted from peripheral blood leukocytes of moderate (MOD, n = 11) and severe (SEV, n = 15) anaphylaxis patients at the time of enrolment in the emergency department (ED) (T0), and again after one (T1) and 3 (T3) hours. Gene expression data are presented as calibrated normalized relative quantities (CNRQ). Boxplots identify the median, upper and lower quartiles, and the range. Dotted lines indicate the median CNRQ of healthy controls (n = 20). Due to the number and complexity of comparisons, significant differences between groups are described in the results text and in Table 2.

Fig. 2

Protein concentrations during anaphylaxis. a MMP9 and (b) soluble TREM1 in serum from moderate (n = 33) and severe (n = 36) anaphylaxis patients at the time of admission to the emergency department (ED; T0), and again after one (T1) and 3 (T3) hours. Boxplots identify the median, upper and lower quartiles, and the range. Dotted lines indicate the median concentration of healthy controls (n = 20): 304.1 ng/mL MMP9; and 182.3 pg/mL TREM1. Due to the number and complexity of comparisons, significant differences between groups are described in the results text.