Modulating effects of hexapeptide on the altered phenotype of neutrophil granulocyte CD16+CD62L+CD11b+CD63- and CD16+CD62L+CD11b+CD63+ subsets in children with acute osteomyelitis in the in vitro system

The problem of acute osteomyelitis in children is of special importance among the inflammatory diseases of musculoskeletal system, due to infectious conditions arising in the body and spreading to the bone tissue caused by impaired immune regulation, first of all, concerning neutrophilic granulocytes. Of interest is studying the subsets of neutrophilic granulocytes arising when the cells are involved into the inflammatory process in acute pediatric osteomyelitis, and determining the opportinity to influence the level of receptor expression aiming for correction of their functions. The purpose of our study was to evaluate the effect of hexapeptide arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine on the altered phenotype of neutrophilic granulocytes in children with acute osteomyelitis using an in vitro experimental model. We examined the peripheral blood samples from children with acute hematogenous or post-traumatic osteomyelitis at the age of 10 to 17 years (n = 12) upon their admission to the hospital, and from healthy children (n = 7). Blood samples from children with acute osteomyelitis were incubated with hexapeptide (10^-6 g/L) for 60 min, at 37 °C. The content of neutrophilic granulocyte subsets (CD16⁺CD62L⁺CD11b⁺CD63^- and CD16⁺CD62L⁺CD11b⁺CD63⁺), expression density of appropriate membrane receptors were assessed by flow cytometric technique (FC 500 “Beckman Coulter”, USA). Phagocytic function was studied by assessing the degree of completed phagocytosis of S. aureus. It was found that, in acute osteomyelitis, a 8.5-fold increased proportion of activated CD16⁺CD62L⁺CD11b⁺CD63⁺NG subset with the CD16^brightCD62L^brightCD11b^brightCD63^dimNG phenotype was revealed, along with a decrease in the CD16⁺CD62L⁺CD11b⁺CD63^-NG subset and changes in the CD16^dimCD62L^brightCD11b^midCD63^- NG phenotype as compared with reference indexes of healthy children. At the same time, an increased number of actively phagocytic cells was noted, however, with decreased indexes characterizing capture and digestion of the bacterial antigen. In the in vitro experiments, the tested hexapeptide was shown to modulate the phenotypes of both studied subsets (CD16^brightCD62L^midCD11b^midCD63^- and CD16^midCD62L^midCD11b^midCD63^dimNG), thus promoting restoration of the receptor expression levels to the reference group values, as well as phagocytic activity, in terms of uptake and digestive capacity of microbial cells. Thus, the dominance of a diagnostically significant activated CD16⁺CD62L⁺CD11b⁺CD63⁺ neutrophil subset with the CD16^brightCD62L^brightCD11b^brightCD63^dimNG phenotype was found in acute osteomyelitis in children. The results of in vitro studies have shown that the hexapeptide caused phenotypic modulation of the CD16⁺CD62L⁺CD11b⁺CD63^- neutrophils, and CD16⁺CD62L⁺CD11b⁺CD63⁺NG subsets, along with recovery of their phagocytic activity. In the future, our results may provide a basis for the development of new effective therapeutic regimens.