Increased eosinophil colony formation in agar by haemopoietic cells from patients with the hypereosinophilic syndrome

Summary Colony formation by granulocyte/macrophage and eosinophilic progenitor cells in the blood and/or bone marrow of four patients with ‘hypereosinophilic syndrome’ (HES) was studied in agar culture. Colony-stimulating activity (CSA) was derived from leucocyte feeder layers (LFL) or from medium conditioned by phytohaemagglutinin-stimulated lymphocytes (LCM). The total numbers of colonies in the patients' blood and marrow were normal. When patients' marrow cells were cultured over LFL, the proportion of colonies that were eosinophilic was greater than normal (mean 21% vs 6%, P < 0.001) and this difference was accentuated when CSA was derived from LCM (mean 53% vs 15%, P < 0.001). LCM derived from normal subjects and LCM prepared from HES patients had similar effects. In the blood the total number of colonies and the proportion of eosinophilic colonies were similar in patients and controls. The overall pattern of colony formation HES differed distinctly from that observed in a patient with eosinophilic leukaemia reported previously. We conclude that the proportion of eosinophil-committed progenitor cells in the marrow of patients with HES is increased, but we have failed to demonstrate a role for the patient's own lymphocytes in augmenting eosinophil production.     

Inhibition of CFU-NM and CFU-EOS by mature granulocytes

Approximately half of the colony-forming units-culture (CFU-C) from normal peripheral blood are eosinophilic. The purpose of our study was to determine: (1) whether progenitor cells committed to eosinophil or neutrophil maturation would be differentially affected by feedback inhibition, and (2) whether mature eosinophils added to the feeder layers of the culture would inhibit the proliferation of CFU-C in a manner similar to that described for neutrophils. Concentrated eosinophils and neutrophils, obtained by separation on a metrizamide gradient, were added to feeder layers containing either 10(6) autologous whole mononuclear cells (WMNC) or 0.1 ml of leukocyte conditioned media (LCM). The average number of colonies was 123/10(6) nonadherent cells (NAC) cultured. When neutrophils or eosinophils were added to the WMNC feeder layer, the percent inhibition of growth was 40.2% +/- 1.6% (mean +/- SEM) and 42.3% +/- 5.4%, respectively, but the ratio of neutrophil to eosinophil colonies remained constant. No effect was seen when neutrophils or eosinophils were added to an LCM feeder layer. Thus, it appears that the differential control of neutrophil versus eosinophil production in vitro is not regulated through feedback inhibition by mature granulocytes. In addition, these studies suggest that eosinophils, as well as neutrophils, cause inhibition of CFU-C growth when intact cells are the source of colony-stimulating factor (CSF).     

Cyclosporin for hypereosinophilic syndrome

Summary The idiopathic hypereosinophilic syndrome (HES) comprises a heterogeneous group of disorders with unknown pathogenesis characterized by persistent peripheral blood and bone marrow eosinophilia and eosinophil infiltrates of multiple organs, leading to severe organ dysfunction. Lymphokine-mediated T-lymphocyte control of human eosinophilic granulopoiesis is thought to play a major role in the pathogenesis of HES. Treatment of this disease with cyclosporin-A (CSA) therefore appears to be sensible. We report the case of a patient with a severe HES who failed to respond adequately to glucocorticoid treatment. With additional CSA therapy, disease activity showed a favorable remission and eosinophil counts rapidly decreased to the normal range and remained normal even after reduction of the methylprednisolone dosage to 7.5 mg daily. To date his remission has continued for more than 6 months. In the meantime we have confirmed the beneficial effects of CSA in two other cases of HES. This suggests that CSA treatment is justified, at least in combination with glucocorticoids, in severe cases of HES to prevent the side effects of a longlasting high-dose glucocorticoid therapy.     

Elevated serum levels of soluble interleukin-2 receptor: a marker of disease activity in the hypereosinophilic syndrome.

We report here the presence of very high serum levels of the soluble interleukin-2 receptor (sIL-2R) in patients with blood hypereosinophilia with or without detectable markers of malignancy or signs of visceral involvement. The highest sIL-2R levels were observed in 16 eosinophilic patients with T-cell lymphoma (3,440 to 79,500 U/mL). Elevated levels of sIL-2R were also present (1,330 to 22,500 U/mL) in sera from 38 patients with the hypereosinophilic syndrome (HES) without detectable T-cell lymphoma. In this group of patients, the highest levels were noted in the patients with the malignant form of HES. Significantly lower levels were measured in sera of patients with hypereosinophilia associated with parasitic diseases, allergic disorders, or other miscellaneous diseases. Elevated serum sIL-2R levels were not closely paralleled by changes in the number of CD25-positive peripheral blood mononuclear cells as assessed by flow cytometric analysis. However, expression of IL-2R messenger RNA was detected in blood mononuclear cells collected from HES patients. In eight eosinophilic patients with T-cell lymphoma, the serum sIL-2R levels were significantly correlated with the eosinophil counts, and with the total number of blood hypodense eosinophils. alpha-Interferon (alpha-IFN) therapy resulted in both a dramatic clinical improvement and a rapid decrease in sIL-2R levels and blood hypereosinophilia. Similar beneficial effects of alpha-IFN were noted in patients with malignant HES who lacked a detectable T-cell lymphoma. Our data indicate that HES is associated with elevated serum IL-2R levels. The highest levels were observed in the most severe forms of HES with hematologic markers of malignancy or evident visceral involvement. Serum levels of sIL-2R might represent a useful indicator for the management of HES patients. In addition, the respective changes of sIL-2R and blood eosinophilia might reflect distinct processes of mononuclear cell activation affecting the eosinophil lineage.     

Imatinib therapy for hypereosinophilic syndrome and other eosinophilic disorders

Imatinib mesylate (Gleevec), a small molecule inhibitor of abl, kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, has been reported to be effective in the treatment of hypereosinophilic syndrome (HES) and a rare eosinophilia-associated chronic myeloid disorder (eos-CMD) characterized by the t(5;12)(q33;p13) cytogenetic abnormality. In the current study, we sought to confirm the preliminary observations in HES as well as evaluate the therapeutic value of imatinib in eos-CMD that is not associated with t(5;12)(q33;p13). Five patients with HES (all men, median age = 46 years) and 2 with eos-CMD (both men, aged 45 and 58 years) were treated with imatinib at a starting dose of 100 to 400 mg/day. Cytogenetic studies showed no evidence of either the bcr-abl translocation or t(5;12)(q33;p13) in any patient. Screening of exons encoding the intracellular catalytic domains and extracellular ligand binding domains of PDGFR beta (exons 2-23) and c-kit (exons 1-21) in 6 patients demonstrated mostly previously known polymorphisms. At a median follow-up of 17 weeks (range, 10-33 weeks), 2 patients with HES and 1 with eos-CMD have achieved complete clinical remission and 1 additional patient with HES has achieved a partial remission. In contrast to previous observations, all 4 responding patients had elevated serum interleukin-5 levels. Although the drug was well tolerated in most patients, a previously unrecognized treatment toxicity of acute left ventricular dysfunction occurred in a responding patient with HES within the first week of treatment. Myocardial biopsy revealed eosinophilic infiltration and degranulation, and the cardiogenic shock was reversed with the prompt institution of corticosteroid therapy.     

Functional Cellular Maturation in Cultures of Human Haematopoietic Cells

S ummary . The capacity of cells grown in colonies derived from human haematopoietic cells to phagocytize bacteria and to reduce nitroblue tetrazolium (NBT) was studied as a measurement of functional maturation. Marrow cells from 12 non-leukaemic patients, fivc patients with acute non-lymphocytic leukacmia, and cells taken from a myeloblastic cell line established from a patient with acute myelo-genous leukaemia (AML) were studied. Cells were cultured in soft agar with normal human leucocyte feeder layers as the source of stimulating factor. While the leukaemic marrows gave rise to fewer colonies than the non-leukaemic marrows, colony formation by the AML cell line was extensive. In all instances colony formation was completely dependent upon the presence of the leucocyte feeder layers. Suspensions of pooled colonies contained 42% mature granulocytes in non-leukaemic cultures and 41% in the leukaemic cultures. Granulocytes from these cultures showed active phagocytosis of Staphylococcus aureus and reduction of NBT. In cells taken directly from the liquid suspension culture of the AML cell line, no mature granulocytes nor phagocytosis were noted, whereas 35% and 26% of the cells removed from the colonies formed by these cells were classified as mature granulocytes, or demonstrated phagocytosis and NBT reduction respectively. These observations indicate that granulocytes in soft agar colonies derived from human haematopoietic cells are functionally mature, and that cells derivcd from a human AML cell line have the capacity to form colonies with normal maturation, if they are provided with an appropriate stimulating factor.     

Types of colonies formed by normal human bone marrow, peripheral blood and umbilical cord blood CFUc

Four types of colonies can be formed by progenitor cells (CFUc) in double layer agar cultures: eosinophil, granulocyte, macrophage, and mixed granulocyte-macrophage. We have developed a method for in situ staining of intact agar plates with luxol fast blue and acetoorcein employed to differentiate the type of colonies. Cultures of 10(5) normal human bone marrow cells yielded 89.1 colonies, of which 72% were eosinophil. Cultures of 5 X 10(5) peripheral blood cells, enriched by isopycnic sedimentation, yielded 39.0 colonies, of which 48% were eosinophil. T-cell depleted peripheral blood mononuclear cells yielded 116.9 colonies/5 X 10(5) cells, of which 74% were eosinophil. Umbilical cord blood cells yielded 65.1 colonies/5 X 10(5) cells, of which 23% were eosinophil. Comparing similar numbers of total colonies, there appears to be fewer relative and absolute numbers of eosinophil colonies formed by CFUc recovered from cord blood as opposed to adult peripheral blood and bone marrow. Enumeration of colony numbers without regard to types of colonies is imprecise. The method employed in the present study is easy to perform, reproducible and provides permanent slides.     

Effect of High Dose Busulphan on Leukaemic Progenitor Cells in Chronic Myeloid Leukaemia

Abstract: Six patients with Philadelphia positive chronic myeloid leukaemia (CML) were treated with single high doses of busulphan .
The action of busulphan on the in vivo kinetics of circulating progenitor cells (colony forming cells) was measured using an agar culture system which involved scoring of total colonies and clusters at 7 days and of granulocyte, monocyte and eosinophil colonies at 14 days .
High dose busulphan was found to be effective in suppressing circulating granulocyte, monocyte and eosinophil progenitor cells. The effect of busulphan on progenitor cells was rapid and their levels fell by at least 83% within five days. By contrast, the white blood cells fell by only 9% and the platelets fell by 10% over this time. Subsequently, the white cell count and platelet count fell to near normal levels. The progenitor cell levels began to rise again at a mean of 35 days following busulphan treatment and the white blood cells at a mean of 39 days in four patients. One patient remained in haematological remission for six months following 100 mg of busulphan with less than 1 progenitor per 5 × 10⁵ peripheral blood nucleated cells. One patient in myeloblastic transformation had a previously not described culture pattern consisting of a high cluster to colony ratio at 7 days and of an increased number of predominantly eosinophilic colonies at 14 days. There was no significant fall in progenitor cell levels following busulphan and this patient died.     

Multilineage involvement in hypereosinophilic syndrome terminating in granulocytic sarcoma and leukaemic transformation with trisomy 8

We report a patient with hypereosinophilic syndrome (HES), which, 8 years later, transformed into granulocytic sarcoma in the brain and, subsequently, into acute myelocytic leukaemia. Repeated chromosome analyses showed a normal karyotype, until the time of leukaemic transformation when trisomy 8 was confirmed in cells from the bone marrow and cerebrospinal fluid. The combined techniques of May-Grunwald-Giemsa staining and fluorescence in situ hybridization identified trisomy 8 not only in blasts and eosinophils but also in neutrophils and erythroblasts. Our observation suggests that HES is a multilineage myeloproliferative disorder involving precursors of at least the eosinophil, neutrophil and erythroid lineages.     

Interferon treatment for hypereosinophilic syndromes and systemic mastocytosis

Hypereosinophilic syndromes (HES) and systemic mastocytosis (SMCD) are heterogeneous disorders with clinical symptoms from local and remote effects of excessive proliferation of eosinophils and mast cells, respectively. Interferon alpha (IFN-alpha), alone or in combination with other medications, can be a useful, and at times life-saving, treatment for patients with HES. Receptors for IFN-alpha are present on eosinophils, and clinical benefits are due to its effect on eosinophil proliferation, migration, activation, and survival. These effects are likely mediated through multiple pathways including, but not limited to, inhibition of eosinophil colony-forming cells, upregulation of IFN-gamma synthesis, and inhibition of production of eosinophil-active cytokines by T cells, mast cells, and mononuclear cells. IFN-alpha has been life-saving for patients with intractable HES that were resistant to prednisone, hydroxyurea, and other agents. Resistance to the eosinopenic effect of IFN-alpha does not develop and the dose of IFN-alpha necessary to maintain control of eosinophilia often decreases with time. The combination of IFN-alpha and hydroxyurea is very useful and allows dosage reduction of IFN-alpha and better control of hypereosinophilia than with either agent alone. The efficacy of IFN-alpha for treatment of SMCD has been more difficult to establish, with both favorable and unfavorable results reported. The disparate results may have resulted from the small number of patients with SMCD treated with IFN-alpha, the use of various criteria for a "successful" treatment outcome, short duration of treatment and follow-up, and the use of modest dosages. In reported series, side effects from IFN-alpha have frequently been dose-limiting. IFN-alpha improves many of the clinical symptoms of SMCD including dermatological, hematological, gastrointestinal, and systemic symptoms associated with histamine release. IFN-alpha has a beneficial effect on skeletal symptoms because of its ability to increase bone density and reduce painful episodes from vertebral fractures. No consistent improvement in bone marrow infiltration by mast cells has been demonstrated except in a recent study employing high dosages of IFN-alpha. A beneficial effect from the combination of IFN-alpha and prednisone has been reported for several patients, suggesting that combined use of these two medications may provide synergism in treatment outcomes.     

Cytogenetic and molecular cytogenetic characterization of 6 new cases of idiopathic hypereosinophilic syndrome

BACKGROUND AND OBJECTIVE: Idiopathic hypereosinophilic syndrome (HES) is defined as a peripheral blood eosiniphilia greater than 1, 500 cells/microL for longer than 6 months, absence of other apparent etiologies for eosinophilia and signs and symptoms of organ involvement. HES may be a reactive condition or a chronic myeloproliferative disorder but scanty information is available concerning its cytogenetic profile. DESIGN AND METHODS: Six patients with HES were studied by cytogenetic analysis. To increase the sensitivity of cytogenetic analysis, interphase FISH studies were performed to detect some cryptic chromosomal lesions involving the regions known to be frequently involved in myeloproliferative disorders (i.e. BCR/ABL, 5q31, 7q31.1, 11q23, 13q14, 17p13). Clinical parameters were recorded in all patients. RESULTS: A 3q deletion was detected in one patient; two unrelated clones with +14 and +11 were present in another patient who had a cryptic 5q31 deletion as disclosed by FISH; both patients had a mild clinical course. The 5q31 deletion was shown to involve the eosinophilic lineage and not the lymphoid cells. No chromosome abnormalities were found by karyotyping or interphase FISH in the remaining 4 cases. In two of these cases the clinical course was aggressive, with progressive leukocytosis and marked splenomegaly in one patient, central nervous system and cardiac involvement as well as bone marrow failure in the other. INTERPRETATION AND CONCLUSIONS: The 3q deletion, +11 and +14, and a cryptic 5q31 deletion involving the cells of the eosinophilic lineage are three novel chromosome abnormalities occurring in HES. We did not find a correlation between evolving or aggressive disease and the presence of chromosome anomalies. Our data confirm that HES is a clinically and biologically heterogeneous condition and suggest that more cases need to be studied to identify clinically significant chromosome changes in this rare condition. Some patients may benefit from treatment with interferon.     

Decreased number of circulating BFU-Es in paroxysmal nocturnal hemoglobinuria

In order to quantitate early erythroid progenitor cells in paroxysmal nocturnal hemoglobinuria (PNH), we have cultured peripheral blood mononuclear cells from 7 PNH patients in a 0.8% methylcellulose medium containing erythropoietin, 2 U/ml. In our experimental conditions, the number of erythroid colonies obtained per 5 X 10(5) mononuclear cells plated was 20.1 +/- 1.9 (SEM) in normal subjects and 2.8 +/- 0.56 (SEM) in PNH patients. In plates from PNH subjects, 38 of 117 showed no growth of erythroid colonies, whereas plates from normal subjects always had colonies. Our findings suggest that PNH patients, despite their hemolytic condition, have a depleted erythroid precursor compartment, and this may play a major role in the pathogenesis of their anemia.     

Allogeneic peripheral blood cell transplantation for hypereosinophilic syndrome with myelofibrosis

Patients with hypereosinophilic syndrome (HES) display a very heterogeneous clinical picture ranging from asymptomatic cases to very aggressive forms. We report a 38-year-old woman with progressive HES who developed severe myelofibrosis and was treated by allogeneic stem cell transplantation, using peripheral blood (PBSCT) instead of bone marrow as the source of progenitor cells, after conditioning with cytoxan and busulphan. To the best of our knowledge, this is the first case of HES with myelofibrosis treated with PBSCT. The patient remains alive 8 months post-PBSCT, and bone marrow fibrosis has significantly decreased following transplantation. Bone Marrow Transplantation (2000) 25, 217-218.     

Interaction between normal and leukaemic human cells in agar culture.

The influence of leukaemic cells from 12 patients with acute leukaemia on normal granulopoiesis in agar culture was investigated using leukeamic cell feeder layers. Leukaemic feeder cells from 7 of the 12 patients elicited no colony growth, while cells from the remaining 5 stimulated normal colony growth. In 3 of the 7 non-stimulatory patients release of inhibitory factors from the leukaemic cells seemed responsible for the effect on normal granulopoiesis, while inappropriate colony stimulating factor (CSF) production by the feeder cells could not be ruled out in the remaining 4 patients. When the leukaemic cells were cultured with, as well as without, conditioned medium, cells from 5 of the patients formed clusters. Growth in these cultures did not correlate to the effect found in the feeder layer experiments.     

Effective treatment of hypereosinophilic syndrome with imatinib mesylate

Imatinib mesylate treatment is highly effective in chronic myeloid leukaemia and recent data have suggested that imatinib mesylate is also effective in the treatment of idiopathic hypereosinophilic syndrome (HES). Six patients with HES were treated daily with 100 mg imatinib mesylate. Five patients had normal karyotype and one showed trisomy 8. RT-PCR was negative for ETV6-PDGFRB and BCR-ABL fusion mRNAs. All patients rapidly achieved complete haematological remission. One patient remained in remission for more than 6 weeks after discontinuing treatment. No significant side effect was noted. Imatinib mesylate should be considered in the first-line therapy of idiopathic HES.     

Possible mechanisms of fibrin deposition in the hypereosinophilic syndrome

Patients with the hypereosinophilic syndrome (HES) are at increased risk of thrombosis and have signs of fibrin deposition in the myocardial cavity; the pathogenesis of these complications is still unknown. We have studied a 51-year-old man affected by HES with heart, lung, skin, and gastrointestinal involvement. Routine laboratory parameters of the hemostatic system were normal with the exception of blood fibrinolytic activity. The latter was evaluated by both diluted blood clot lysis time and euglobulin lytic activity on fibrin plates before and after 10 min venous occlusion. The fibrinolytic activity measured on four occasions during a 3-month period, was impaired both in basal conditions and following venous occlusion. Platelet studies on two different occasions before and during therapy showed spontaneous platelet aggregation, lowered threshold concentrations of various aggregating agents, reduced platelet regeneration time and increased plasma beta-thromboglobulin concentration. The patient's polymorphonuclear cells (more than 75% eosinophils) were devoid of any procoagulant activity (PCA). Instead, patient's mononuclear cells studied before therapy generated significantly higher PCA on stimulation by endotoxin than cells from control subjects. The procoagulant response to endotoxin decreased markedly during therapy. The observed abnormalities could, at least partially, contribute to fibrin deposition in HES.     

Treatment of hypereosinophilic syndrome with imatinib mesilate

Patients with hypereosinophilic syndrome show persistent eosinophilia without recognised cause. We treated five such patients with 100 mg imatinib mesilate (formerly STI-571) daily; four male patients with normal serum interleukin 5 showed complete haematological responses; a female patient who did not respond had raised serum interleukin-5 concentrations. One patient developed leucopenia after 4 days of treatment; counts returned to normal when treatment was stopped. After 1 month, eosinophilia recurred; with further treatment for 2 days, eosinophil counts again became normal. All patients who responded stopped other treatments and reduced imatinib mesilate to 200 mg per week. This drug effectively controls eosinophilia in patients with hypereosinophilic syndrome and normal interleukin-5 concentrations.     

Clonal Th2 lymphocytes in patients with the idiopathic hypereosinophilic syndrome

Idiopathic hypereosinophilic syndrome (HES) and Gleich's syndrome are related disorders characterized by persistent or recurrent hypereosinophilia of unknown origin. Elevated IgE levels and polyclonal hypergammaglobulinaemia are considered as markers of benign outcome in this setting as they are generally associated with predominant cutaneous manifestations and favourable response to glucocorticoid therapy. In a previous study, we identified a clonal population of CD3-CD4+ Th2-like lymphocytes secreting interleukin (IL)-5 and IL-4 in peripheral blood of a patient fulfilling the diagnostic criteria of HES with associated serum hyper-IgE. We now extend this observation by describing identical findings in three additional patients, and we compare their clinical and biological parameters with five other patients with HES. Chromosomal abnormalities were detected in purified CD3-CD4+ Th2 cells from three patients, among whom one developed anaplastic null cell lymphoma. We therefore suggest that a careful search for T-lymphocyte clonality and cytogenetic changes should be included in the work-up of HES for adequate management.     

Obstructive prosthetic mitral valve thrombosis in idiopathic hypereosinophilic syndrome: a case report and review of the literature

Idiopathic hypereosinophilic syndrome (HES) is an uncommon condition characterized by an unexplained elevation of absolute eosinophil count (AEC) to > or = 1.5 x 10(9)/l for at least six months, and is frequently associated with eosinophil-mediated end organ damage. Idiopathic HES, as with secondary HES and primary hypereosinophilic clonal hematopoietic disorders, has a high incidence of myocardial, pulmonary, neurological and other organ injury. Myocardial fibroelastosis and valvular lesions are common, and successful treatment with valve replacement or resection of fibrotic myocardium has been reported. The case is described of a patient with idiopathic HES and multi-organ complications including severe mitral valve disease, in whom a functionally obstructive thrombosis of a newly inserted prosthetic mitral valve occurred despite adequate anticoagulation, when the AEC was profoundly elevated. Recurrent thrombosis has not occurred over a substantial period following AEC reduction with corticosteroids, and subsequent maintenance at normal levels.