Jtm Dewolf

The effect of cyclosporine on haematological parameters in patients with paroxysmal nocturnal haemoglobinuria

Four patients with paroxysmal nocturnal haemoglobinuria (PNH) were treated with cyclosporine. The treatment with cyclosporine was based on the hypothesis that immune-mediated bone-marrow damage is the common pathogenetic mechanism of aplasia and PNH, with lack of GPI-linked ligands for an immune attack (i.e. LFA-3, CD58) rendering PNH cells a growth advantage over other bone marrow cells. In the first patient, presenting with a mixed AA/PNH syndrome, a gradual recovery from aplasia was seen after prolonged treatment with cyclosporine. In a second patient, with a mixed AA/PNH syndrome, no haematological improvement was noted during cyclosporine administration, but this patient became transfusion-independent with increasing neutrophil and platelet counts after a course of ATG in combination with androgen therapy. Both these patients showed an increment in the proportion of neutrophils with normal expression of GPI-linked proteins concurrently with the improvement of haematological characteristics. In the two other patients, presenting with typical PNH, cyclosporine treatment did not result in any change in haematological characteristics, nor in PNH parameters. No significant change in haemolytic parameters was seen in any of the patients. It is concluded that immunosuppressive therapy may be of benefit in patients with a mixed AA/PNH syndrome. This effect became apparent after prolonged treatment with cyclosporine in one patient, and after a subsequent course of ATG with concomitant androgen therapy in another.

Interleukin-4 prevents the induction of G-CSF mRNA in human adherent monocytes in response to endotoxin and IL-1 stimulation

Human recombinant interleukin‐4 (IL‐4) was studied for its effects on the expression of granulocyte‐colony stimulating factor (G‐CSF) mRNA in human adherent monocytes in the absence and presence of endotoxin and interleukin 1 (IL‐1), IL‐4 (15 ng/ml) did not induce G‐CSF transcripts in monocytes but suppressed the endotoxin‐induced G‐CSF expression when added simultaneously. Sequential treatment of monocytes with IL‐4 followed by endotoxin suppressed G‐CSF mRNA induction totally. This effect was independent of the presence of fetal bovine serum but dependent of the IL‐4 dose. Comparable results were obtained with IL‐1, IL‐1 (50 U/ml) induced G‐CSF expression in human adherent monocytes which could be counteracted by IL‐4 pretreatment. In addition, it was shown that the induction of G‐CSF mRNA by the calcium‐ionophore A23187 or by c‐AMP elevating agents could be blocked by IL‐4. These suppressive effects of IL‐4 were not related to changes in the half‐life of G‐CSF mRNA and were independent of protein synthesis. Finally it was demonstrated that IL‐4 had comparable effects on the G‐CSF secretion of endotoxin and IL‐1 stimulated human monocytes by using a murine bone marrow assay. These results indicate that IL‐4 down‐regulates the expression of G‐CSF gene and secretion of proteins in human activated monocytes.

Correction of neutropenia by GM-CSF in patients with a large granular lymphocyte proliferation.

SummaryThe in vivo and in vitro effects of GM-CSF were tested in four patients with large granular lymphocyte proliferation (LGLP) and severe granulocytopenia. All patients had an increased percentage of LGL cells (> 20%), whereas 3/4 patients demonstrated rearranged T-cell-receptor genes. An effect on the peripheral granulocyte counts was noticed in 3/4 patients after 14 days of GM-CSF administration (5Μg/kg/day, subcutaneously); 2.5, 7, and 0.45×109/1, respectively. These changes were associated with a two- to five-fold increase in monocytes and a strong increment in eosinophils. In one patient GM-CSF was not effective in increasing the granulocyte count. B lymphocytes showed a variable increase, ranging from 1.2-fold to 3.8-fold, while the number of NK cells remained almost constant during the GM-CSF treatment. No consistent effect of GM-CSF on T lymphocytes was observed. These data suggest that GM-CSF may be a therapeutic option in some patients with LGLP complicated by granulocytopenia and/or infection.

INTERLEUKIN-4 SUPPRESSES THE INTERLEUKIN-3 DEPENDENT ERYTHROID COLONY FORMATION FROM NORMAL HUMAN BONE-MARROW CELLS

Human recombinant interleukin 4 (IL‐4) was studied for its effects on the erythroid burst forming unit (BFU‐E) from human bone marrow cells. IL‐4 alone neither supports nor suppresses the erythropoietin (Epo)‐dependent colony formation. Different results were obtained when IL‐4 was combined with interleukin‐3 (IL‐3) in the presence of Epo. IL‐4 suppressed the IL‐3 supported erythroid colony formation in all cases (an increase of 58 ± 8% with IL‐3 versus an increase of 14 ± 7% with IL‐3 plus IL‐4, n= 8). This antagonizing effect was dependent on the continuous presence of IL‐4 in the culture medium, but was independent of adherent cells. B‐, T‐cells, or the presence of serum in the culture medium. Finally, the effects of IL‐4 and IL‐3 were studied on the ‘Epo‐independent’BFU‐E by adding Epo on day 3. A decline of the IL‐3 supported BFU‐E was observed in the presence of IL‐4 but the degree of reduction was equivalent to the results obtained when Epo was supplied at day 0. These findings indicate that IL‐4 acts as suppressive growth factor for the IL‐3 supported erythroid colony formation from human bone marrow cells.

The effects of IL-1 and IL-4 on the Epo-independent erythroid progenitor in polycythaemia vera

Summary. Human recombinant interleukin‐1 (IL‐1) was studied for its effects on the erythroid progenitors from normal subjects and from patients with polycythaemia vera (PV). No supportive effect of IL‐1 was noticed on the normal erythropoietin (Epo) dependent, erythroid burst‐forming unit (BFU‐E) using peripheral blood or bone marrow. In contrast, the Epo‐independent BFU‐E from peripheral blood of PV patients could be stimulated significantly. This enhancing effect of IL‐1 was not only observed with unsorted but also with sorted CD34+ cells. In addition, it was shown that IL‐1 indirectly stimulated the Epo‐independent BFU‐E because anti‐GM‐CSF could abrogate the supportive effects of IL‐1. In contrast to the Epo‐independent BFU‐E, the Epo‐dependent erythroid colony formation from PV patients could not be augmented by IL‐1. Finally, we studied the effects of IL‐4 on the Epo‐independent BFU‐E, because IL‐4 can affect the erythroid colony formation and modulate the effects of IL‐1. IL‐4 suppressed the Epo‐independent BFU‐E. This effect could be counteracted by the addition of IL‐1 to the culture medium. However, the suppressive effect of IL‐4 was not related to a decline in spontaneous release of IL‐1, because an anti‐IL‐1 antibody did not modify the spontaneous erythroid colony formation. These data indicate that IL‐1 and IL‐4 exert separate influences on the Epo‐independent erythroid colony formation in PV.

SUCCESSFUL IMMUNOSUPPRESSIVE TREATMENT AFTER FAILURE OF ERYTHROPOIETIN THERAPY IN TWO SUBJECTS WITH REFRACTORY ANAEMIA

The management of refractory anaemia (RA), one of the myelodysplastic syndromes (MDS), is mainly by red cell transfusions. Other forms of treatment, such as androgens, cytotoxic agents, ‘differentiation-inducing’ drugs or haematopoietic growth factors have generally shown disappointing results (Cheson, 1990; Schouten et al, 1991). We present data on two patients with a.typica1 RA, who were treated with recombinant human erythropoietin (Epo) unsuccessfully and subsequently with immunosuppressive agents resulting in almost complete haematological recovery. Patient 1, born in 19 5 1, presented with amaemia in 19 8 5 . He was transfusion dependent since that time. In 1989 he was referred to our hospital: he was not using medication at the time. Full blood count showed haemoglobin 9.5 g/dl, MCV 8 5 8, reticulocytes 0%. platelets 100 x 109/1, leucocytes 2.7 x 10y/l with 1% eosinophils, 1% basopliils, 42% neutrophils, 53% lymphocytes and 3% monocytes. Bone marrow cytologic and histologic examination showed hypercellularity with increased erythropoiesis. many dysplastic forms, active granulopoiesis, megakaryocytes with abnormal nuclear morphology and less than 5% blasts. No chromosomal abnormality was detected. A diagnosis of RA was made after other causes of anaemia had been extensively excluded. In vitro erythroid colony formation (burst forming unitserythroid, BFU-E) from bone marrow yielded 108 BFU-E/lOS mononuclear cells, not significantly different from control values (146 f 39). In 1990 he was treated with Epo subcutaneously or intravenously at doses up to 320 U/kg three times weekly for 6 weeks. No beneficial effect on transfusion frequency was observed (Fig 1). Subsequently, prednisolone (60 mg/d) was started. This resulted in an improvement in the haemoglobin concentration. Later, the prednisolone was