High Incidence of Transiently Appearing Complement-Sensitive Bone Marrow Precursor Cells in Patients with Severe Aplastic Anemia - A Possible Role of High Endogenous IL-2 in Their Suppression

Abstract

In a prospective long-term study on the incidence of paroxysmal nocturnal hemoglobinuria (PNH), 115 consecutive patients with severe aplastic anemia (SAA), 97 treated with antilymphocyte globulin (ALG) and 18 with bone marrow transplantation (BMT), were observed over a period of 4–18 years and tested for the presence of complement-sensitive hematopoietic precursor cells with the bone marrow (BM) sucrose test. Sixteen (14%) of the ALG-treated patients developed clinical signs of PNH between 0.5 and 8 years after treatment. Complement-sensitive BM precursors were found in 89% of the SAA patients at some time during their disease, but in none of 18 normal donors. At diagnosis, their proportion was significantly higher in patients who later developed PNH than in patients who later achieved disease-free complete remission (CR). After ALG, the abnormal population was found in both groups, but it was gradually replaced by normal precursors in remission patients. After BMT, the complement-sensitive population decreased to very low numbers in patients with a stable graft, but increased again in 3 patients upon graft rejection. Mimicking the PNH defect by enzymatic removal of glycosyl-phosphatidylinositol (GPI)-linked proteins from CD34+ cells resulted in their complement sensitivity, suggesting that the BM sucrose test identifies precursor cells carrying the PNH defect. In 66 patients, white blood cells (WBC) in peripheral blood (PB) were examined for GPI-deficient populations by flow cytometry (FACS). Ten patients with signs of clinical or laboratory PNH had over 25% complement-sensitive precursor cells in the BM and a GPI-deficient WBC population in the PB. Of 56 SAA patients without PNH, 8 had an abnormal population detectable with both tests, 26 only with the BM sucrose test, 4 only with PB FACS analysis, and in 18, no abnormal cells were detected with either test. In search for parameters which might explain why in some patients the abnormal population expands, while it regresses or disappears in others, we tested the release of IL-2 as a parameter of immune competence. At diagnosis, IL-2 release was approximately 50% of normal in patients who later developed PNH, while it was double the normal value in patients who later achieved CR. We conclude that the majority of SAA patients transiently harbor complement-sensitive precursor cells in the BM. Patients with more than 25% abnormal BM precursors and low endogenous IL-2 release are at risk of progression to clinical PNH.

Cell Cycling Stress in the Monocyte Line as a Risk Factor for Progression of the Aplastic Anaemia/Paroxysmal Nocturnal Haemoglobinuria Syndrome to Myelodysplastic Syndrome

“…Our previous finding that IL-2 release is low in PNH patients compared to AA patients not developing PNH [25]is in keeping with the role of the immune system in suppressing the PNH clone. The observation that the presence of a GPI-lacking clone before IST does not preclude a response, but that these patients have a high risk of later developing PNH [32]is compatible with our hypothesis: PNH cells present at the time of diagnosis of SAA indicate that the immune system failed to eradicate the abnormal clone, and also allowed its expansion after IST.…”

“…Short-term BM cultures in methylcellulose were performed as described [25]. A human leucocyte-conditioned medium with standard activity was used for stimulation.…”

Paroxysmal Nocturnal Haemoglobinuria: A Replacement of Haematopoietic Tissue?

“…GPI-AP-deficient cells are also present in a substantial proportion of patients with AA [5, 6, 7, 8, 9, 10]. Consistent with these findings Bruno Speck’s group demonstrated that the majority of patients with severe AA (SAA) transiently harbour complement-sensitive precursor cells in the bone marrow [11]. …”

“…In vivo application of the cytotoxic Campath-1H antibody which is directed against the GPI-AP CD52 led to a reversible emergence of GPI-AP-deficient lymphocytes and monocytes [44, 45]. The association of low endogenous IL-2 release with progression of AA to PNH points at the potential importance of immunological mechanisms for the selection of PNH cells in vivo [11]. …”

Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes “Why? Why? Why?”

“…In chimeric knockout mice, PIG-Acells also constitute a minor, static fraction of the marrow or circulating hematopoietic compartments, and PIG-A -and normal cells behave similarly in paired tissue culture assays (12)(13)(14). In most laboratories, PNH patient bone marrow or blood, while often globally deficient in hematopoietic progenitor numbers, has not shown unanticipated outgrowth of either the PIG-A -or normal phenotype cells in colony-forming or long-term culture assays that would indicate a functional difference (15)(16)(17)(18). Only one group has reported a major difference between PIG-A -cells and controls in susceptibility to apoptosis, but this finding has not been reproduced in other laboratories (ref.…”