Ambivalent in vitro effect of immunoregulatory peptides on antigen-presenting subsets of neutrophil granulocytes in purulent inflammatory diseases

Neutrophilic granulocytes (NG) are functioning as regulators of the immune response. Expression of NG molecules HLA-DR and presentation of antigen to T cells is one of their regulatory mechanisms. The NG dysfunction plays a great role in pathogenesis of acute hematogenous osteomyelitis (AHO) in children. An activated, antigen-presenting NG subset (APC) СD66b⁺CD16⁺CD33⁺HLA-DR⁺ was also found in these patients. Therefore, studies of surface NG membrane receptor expression, including HLA- DR, their regulation by peptides, and influence of the latter factors on correction on NG effector functions are of sufficient interest. Our objective was to evaluate the possibility of in vitro modulating the phenotype of CD66b⁺CD16⁺CD33⁺HLA- DR^-, CD66b⁺CD16⁺CD33⁺HLA-DR⁺ subsets of neutrophilic granulocytes under the influence of hexapeptide (HP) and glucosaminylmuramyl dipeptide (GMDP) in blood cells of children with acute hematogenous osteomyelitis using in vitro experimental tests.

Peripheral blood (PB) of 28 children with AHO aged 8-15 years was studied (the study group). 13 healthy children aged 8-15 years comprised the comparison group. To evaluate the effect of peptides, PB of children with AHO was cultured with HP (10^-6 g/L, 60 min, 37 °С): study group 1, and with GMDP (10^-6 g/L, 60 min, 37 °С) – study group 2. The number of NG CD66b⁺CD16⁺CD33⁺HLA-DR⁺, CD66b⁺CD16⁺CD33⁺HLA- DR^- subsets, receptor expression density (MFI) (FC 500 “Beckman Coulter”, USA), phagocytic activity of NG, before and after cultivation were tested with these peptides.

In children with AHO, a subset of NG CD66b⁺CD16⁺CD33⁺HLA-DR⁺ is registered in 30.2 (16.4-34.9) %; with MFI, HLA-DR it comprised 3.5 (3.3-4.2) %. Under the influence of HP, a decrease of NG-APC and MFI HLA-DR numbers to 1.7 (1.6-2.2) (p_1.2 > 0.05) was revealed, due to binding of HP to HLA-DR (p > 0.05). Under the influence of GMDP, there is a significant increase in MFI CD66b and MFI CD33 receptors (p_1.2 < 0.05) in both subsets; there is an increase in MFI HLA-DR (p > 0.05) in the NG-APC subset. The modulating effects of HP and GMDP on the phenotype of NG CD66b⁺CD16⁺CD33⁺HLA-DR⁺ and CD66b⁺CD16⁺CD33⁺HLA- DR^- subsets may contribute to restoration of the phagocytic function of NG.

We have detected the “long-lived” activated NG subset CD66b⁺CD16⁺CD33⁺HLA-DR⁺ with the properties of APC, that can present antigen to T lymphocytes in PB of children with AHO. However, the important question exists, whether such a transformation will promote or slow down the progression of the purulent-inflammatory process? In this study, we have demonstrated in vitro the ability of two immunotropic peptides (HP, GMDP) to modulate the phenotype of NG-APC subset, thus potentially promoting recovery of the NG effector functions.