The detection and assay of integrins and soluble integrin ligands as an index of granulocyte function and mass after chemotherapy and stem cell tranplantation with haematopoietic growth factor administration

Abstract

Specific interactions between molecules expressed on the surface of leukocytes and cell adhesion molecules on target cells are important determinants of leukocyte function as well as playing a role in cell - cell communication.Four main groups of adhesion molecules are recognized : the integrinreceptor family; the immunoglobulin superfamily; selectins and cadherins. The focus of the present study is on CD 1 lb/CD 18 ( Mac-1), which belongs to integrin family, and its ligand ICAM-1, which belongs to immunoglobulin family. The s tudies which make up the body of this dissertation were designed to investigate^ a ) the interaction of neutrophil adhesion molecule CD 1 lb/CD 18 and its ligands and ( b ) to develop a marker of neutrophil recovery and function after severe chemotherapy induced neutropenia following either intensive but unsupported conventional dose consolidation chemotherapy or high dose chemotherapy plus autologous stern cell transplantation. These latter studies focussed on the soluble intercellular adhesion molecule-1 (sICAM-1) . The results of the studies presented in this dissertation may be summarized as follows: a ) Adhesion o f neutrophils to endothelial cells by a CDllb/CD18~ dependent mechanism In this investigation the focus of interest was to study the adhesion of normal neutrophils to vascular endothelial cells. While the factors including the role of CD 11/CD 18 responsible for the interaction of neutrophils and endothelium had been previously investigated there was still disagreement about the role of CD 1 lb/CD 18. In this study an in-vitro method was developed whereby adhesion to the ECV-304 cell line was used to elucidate factors involved in neutrophil adhesion to vascular- endothelium. The results showed that adhesion of unstimulated neutrophils to endothelial cells is largely mediated by a GDI lb/CD 18-dependent cell : cell interaction, probably through its ligand ICAM-1 on endothelial cells. In addition, the results showed that normal serum contairrs a potent adhesive factor, which promoted this interaction. This factor was identified as complement fragment iC3b. b ) Serum ICAM-1 concentration following conventional dose consolidation chemotherapy for acute myeloid leukemia and after high dose chemotherapy with autologous haematopoietic stem cell rescue The first study had demonstrated an important role of CD 1 lb/CD 18 in neutrophil adhesion. One of the important ligands of CDllb/CD18 is ICAM-1. A soluble form of this ligand has previously been demonstrated. The focus of this study was to elucidate receptor/ligand interactions. In this investigation, serum concentrations of soluble ICAM-1 (sICAM-1) were studied in patients with acute myeloid leukemia (AML) after conventional dose consolidation chemotherapy and in AML and in breast cancer patients following high dose chemotherapy with autologous haematopoietic stem cell transplantation. Investigations were carried out at 3 phases following treatment; during the chemotherapy induced neutropenic phase (neutrophil counts < 0.5 x 109/1); during early recovery(neutrophil counts 0.5 x 109/1 - 1.0 x 109/1); and at recovery from neutropenia(neutrophil count 1.0 x 109/1 - 2.5x1071). Results showed a significant elevation of serum levels of sICAM-1, above normal, in both groups of patients during the neutropenic phase. A further increase of sICAM-1 was found in conventional dose consolidation chemotherapy treated AML patients during the post-neutropenia recovery phases. By contrast, patients who were treated with high dose chemotherapy plus autologous haematopoietic stem cell transplantation showed a normalisation of sICAM-1 concentration during tire postneutropenic recovery phases. These findings suggest that recovery neutrophil function does not coincide with recovery of neutrophil count following intensive but unsupported chemotherapy while there was rapid recovery of neutrophil function occurred among patients who received autologous haematopoietic stem cell support. c ) Expression o f CDllb/CD18 on neutrophils in patients undergoing consolidation chemotherapy for acute myeloid leukemia and in patients undergoing h igh dose chemoth erapy and autologous haematopoietic stem cell transplantation This investigation dealt with the quantitative expression of CD 1 lb/CD 18 on neutrophils by flow cytometry in same population of patients investigated in the previous study . CDllb/CD18 expression on neutrophils during early and full neutrophil recovery was normal in stem cell transplantation supported patients. By contrast CD 1 lb/CD 18 expression was markedly decreased in patients who received chemotherapy without stem cell support. These results together with those of tire early study indicated that neutrophil abnormalities with potentially important functional implications occur after neutrophil count recovery following chemotherapy and that these defects may be ameliorated by haematopoietic stem cell support. In summary, the results presented here provide evidence that 1 ) CD 1 lb/CD 18 mediates the interaction of resting neutrophils to endothelial cells, probably by recognizing ICAM-1 on endothelium. Complement fragment iC3b promotes this interaction. 2 ) recovery of neutrophil function does not coincide with recovery of neutrophil count following intensive chemotherapy. sICAM-1 is considered as a sensitive marker of neutrophil functional recovery.