Experimental in vitro phenotype reprogramming of two subsets of neutrophilic granulocytes in children with acute destructive pneumonia by means of a synthetic hexapeptide

Abstract

Effector dysfunctions of neutrophil granulocytes are often associated with the occurrence of dysregulatory processes in the antibacterial immune defense. Acute destructive pneumonia is a severe purulent-inflammatory disease associated with discordant functions of effector mechanisms of neutrophil granulocytes and emergence of negatively transformed cell subsets. Therefore, the search for new experimental approaches aimed at re-orientation of negatively altered phenotype of distinct subsets of neutrophilic granulocytes in the children with acute destructive pneumonia by means of various immunotropic substances is quite relevant. The aim of the study was to evaluate the modulating effects of synthetic hexapeptide (Arginyl-alpha-Aspartyl-Lysyl-Valyl-Tyrosyl-Arginine) on the contents and phenotype of 2 functionally significant subsets of major (CD16+CD64-CD32+CD11b+) and minor (CD16+CD64+CD32+CD11b+) subpopulations of neutrophils in a closed in vitro experimental system sampled in the children with atypical acute destructive pneumonia. We have examined twenty peripheral blood samples from 10 children with acute destructive pneumonia, and 40 blood samples of 20 healthy children 2-4 years old. Immunophenotyping of neutrophil granulocytes classified in 2 subsets was performed on the basis of expression density of membrane receptors, according to MFI criteria. Phenotypic features of neutrophil granulocyte subsets were evaluated in the in vitro system before and after incubation of peripheral blood with Hexapeptide (10-6 g/L; 37 C, 60 min). In children with acute destructive pneumonia, compared with conditionally healthy children, the following variants of negative transformation of the neutrophil subsets were established: a significant decrease in the ratios of the major subset, i.e., from 98.0 (96.9-98.7) % o 55.8 (35.3-74.8) %, with a decreased CD16 and CD11b density expression according to MFI, and a significantly increase ratio of the minor neutrophil subset: from 1.3 (0.4-1.6) % to 52.6 (41.8-54.9) %, with increased expression of CD11b receptor, and a decrease in CD64 expression. Immunomodulatory effects of Hexapeptide upon neutrophil granulocytes of children with acute destructive pneumonia have been demonstrated in the closed in vitro system showing positive phenotype remodeling of both cell subsets in the absence of significant quantitative changes. Thus, upon treatment with the hexapeptide, we have found a significantly increased expression of activation receptors CD16, CD11b in the major subset, and a significant decrease in their expression for the minor subset to the levels typical to healthy children. At the same time, hexapeptide did not affect the studied subsets of neutrophils from healthy children, except of increased CD64 expression in the minor subset. The obtained data can be used in future to develop new approaches to the targeted immunotherapy aimed at correcting the phenotype of neutrophil granulocyte subsets in acute destructive pneumonia in children.