Expression and regulation of IL-4 receptors on human monocytes and acute myeloblastic leukemic cells.

Evidence is presented that human monocytes and acute myeloblastic leukemic (AML) cells contain both high and low affinity binding sites for interleukin-4 (IL-4). On monocytes 183 +/- 132 high affinity binding sites per cell with a Kd of 60 +/- 29 pM and 1500 +/- 600 low affinity receptors with a Kd of 2.3 +/- 0.4 nM (X +/- S.D., n = 6) could be demonstrated. On AML cells (n = 11) a comparable number and binding affinity of IL-4 receptors were observed (77 +/- 36 high affinity receptors with Kd 72 +/- 31 pM and 2400 +/- 1000 low affinity receptors with Kd of 2.2 +/- 0.7 nM). In addition, no cross-competition was shown between radiolabeled IL-4 and IL-1-alpha, IL-3, IL-6, IL-7, G-CSF, and GM-CSF. Both types of receptors on monocytes as well as on leukemic blasts could be down-modulated in a similar fashion by IL-4 and activators of protein kinase C (PKC), but not by the calcium ionophore A23187. The down-modulation by PKC activators was caused by an increased internalization, degradation and release of radiolabeled IL-4 in the medium. Finally, the functionality of the IL-4 receptors were tested on AML cells with a 3H-thymidine proliferation assay. In 8/11 cases IL-4 affected AML proliferation. These data demonstrate two different binding sites for IL-4 on normal and leukemic cells, which can be modulated by external activation signals in an analogous way.

The effects of IL-1 beta and IL-4 on the proliferation and endogenous secretion of growth factors by acute myeloblastic leukemic cells.

The effects of interleukin-1 beta (IL-1) and IL-4 were studied on the proliferation of acute myeloid leukemia (AML) cells. IL-1 stimulated tritiated thymidine (3H-TdR) uptake of AML cells in 8/12 cases, whereas IL-4 enhanced 3H-TdR uptake in 5/12. Combination of both factors resulted in an additive effect in 6/12 cases which could be abrogated by the addition of anti-granulocyte-macrophage colony stimulating factor (GM-CSF). To study whether IL-1, IL-4, or IL-1 plus IL-4 affects the AML progenitor cell directly or indirectly by the release of endogenous factors, supernatants of stimulated AML cells (n = 6) were analyzed for GM-CSF, IL-6, and tumor necrosis factor-alpha (TNF) production. IL-1 induced the endogenous secretion of GM-CSF, IL-6, and TNF in most cases. In contrast, no secretion of growth factors was induced by IL-4, whereas in 2 cases IL-4 suppressed the IL-1-induced secretion of GM-CSF, TNF, and IL-6. This was associated with a decline in the proliferative response to IL-1 measured in a clonogenic assay. In addition it was shown that exogenous supplied GM-CSF and TNF could raise the suppressive effects of IL-4 on the IL-1-supported proliferation. In summary these data indicate that the IL-4-supported proliferation is not caused by the endogenous secretion of GM-CSF, IL-6, and TNF. Furthermore the suppressive effect of IL-4 on the IL-1-induced proliferation in some cases may be caused by a reduced secretion of GM-CSF, TNF, and IL-6.

Interleukin-4-mediated inhibition of C-Fos mRNA expression: role of the lipoxygenase directed pathway.

Interleukin-4 inhibits several monocyte functions like A23187-induced expression of cytokines and c-fos and c-jun proto-oncogene mRNA expression. In an attempt to elucidate the mechanism by which this inhibitive effect is mediated, we compared the effect of IL-4 on A23187-induced c-fos and c-jun mRNA expression in conjunction with inhibitors that selectively inhibit the cyclooxygenase dependent (indomethacin) and lipoxygenase dependent (NDGA) pathway of arachidonic acid (AA) metabolism. NDGA inhibited A23187-induced c-fos mRNA expression by a similar magnitude as IL-4, whereas the effect of indomethacin was only minor. A23187-induced c-jun mRNA expression was not affected by indomethacin and only slightly inhibited by NDGA. These results indicate that in human monocytes c-fos mRNA expression is at least partly controlled by the lipoxygenase directed pathway of AA metabolism, whereas the cyclooxygenase dependent pathway is not involved in the regulation of proto-oncogene expression. This was supported by the finding that leukotriene B4 (LTB4) and 5'-hydroperoxyeicosatetraenoic acid (5'-HPTETE), which are two lipoxygenase metabolites, strongly induced c-fos mRNA, whereas c-jun mRNA expression was slightly affected. However, the inhibitive effect of IL-4 could not be ascribed to a reduced production of LTB4 suggesting that the mode of IL-4 action lies behind the conversion of AA to 5'-HPETE and LTB4.

A population-based case-cohort study of drug-associated agranulocytosis.

BACKGROUND: Agranulocytosis is a life-threatening disorder, often caused by drugs. Incidences or risks of drug-induced agranulocytosis are not well known, since it is rare. METHODS: To determine the risk of drug-associated agranulocytosis as a reason for admission to Dutch hospitals, we performed a population-based case-cohort study. Hospital discharge data came from the Dutch Centre for Health Care Information, Utrecht, which contains data on all general and university hospitals in the Netherlands. The reference cohort consisted of all persons in the catchment area of the Pharmaco Morbidity Record Linkage System (PHARMO RLS) in the Netherlands, composing a population of approximately 220 000 to 484 000 persons from 1987 through 1990. All admissions during that period with agranulocytosis or related diagnoses were included in the study (n = 923). The potential causes of agranulocytosis were assessed in all cases classified as probable or possible agranulocytosis. RESULTS: Discharge summaries were received of 753 admissions, of which 678 contained enough information for analysis. Of the 678,108 were classified as "agranulocytosis probable" or as "agranulocytosis possible." In 75 of these 108 cases, agranulocytosis had been the reason for admission. Fifteen patients had used methimazole within 10 days before developing agranulocytosis; 2, carbimazole; 9, sulfasalazine; 8, sulfamethoxazole-trimethoprim; 4, clomipramine hydrochloride; and 2, dipyrone with analgesics, yielding adjusted relative risks of agranulocytosis of 114.8 (for thyroid inhibitors combined) (95% confidence interval [CI], 60.5-218.6), 74.6 (95% CI, 36.3-167.8), 25.1 (95% CI, 11.2-55.0),20.0 (95% CI, 6.1-57.6), and 26.4 (95% CI, 4.4-11.1), respectively. CONCLUSIONS: The highest relative risks were found for thyroid inhibitors, sulfamethoxazole-trimethoprim, sulfasalazine, clomipramine, and dipyrone combined with analgesics.

Technique of labeling monocytes with 111In-Fe colloid for use in humans.

In order to follow monocyte kinetics in hematologic malignancies by means of radioactive labeling, three conditions are necessary: (a) no loss of monocytes during separation, (b) specific labeling of monocytes, and (c) normal functional capacities of the labeled monocytes. In this report a method is described that fulfills the first two conditions and can be executed with maintenance of sterility. Cell labeling was performed using a mononuclear cell suspension consisting of monocytes (20%-50%), lymphocytes (50%-80%), and with minor contamination by granulocytes, thrombocytes, and erythrocytes. This cell suspension was obtained by centrifugation of a leukocyte suspension on a gradient of 16% wt/vol and 22% wt/vol human serum albumin solutions. The recovery of monocytes with this enrichment method approached 100%. Monocytes were labeled by endocytosis of 111In-Fe colloid; monocytes were labeled significantly higher than lymphocytes (P less than 0.001) and granulocytes (P less than 0.01) (Wilcoxon's two-sample test). Cell viability after labeling was greater than 90%.

Interferon-gamma enhances the LPS-induced G-CSF gene expression in human adherent monocytes, which is regulated at transcriptional and posttranscriptional levels.

Human adherent monocytes were studied with regard to the expression of granulocyte colony-stimulating factor (G-CSF) at mRNA and protein levels in response to lipopolysaccharide (LPS) and gamma-interferon (IFN-gamma) stimulation. Monocytes did not express G-CSF transcripts in response to IFN-gamma treatment. In contrast, monocytes exposed to IFN-gamma plus LPS showed a dose-dependent increase in G-CSF mRNA accumulation and protein secretion compared to LPS-stimulated monocytes. The augmented G-CSF mRNA expression in response to IFN-gamma plus LPS was the result of a slight increase in the G-CSF transcription rate (2.2-fold) and a more than 6-fold increase in the G-CSF mRNA half-life (20 minutes vs. > 120 minutes). In addition, it was shown that the effects of IFN-gamma on LPS-induced G-CSF protein secretion could be mimicked by the calcium ionophore A23187, suggesting that the Ca(2+)-dependent pathway might be triggered after binding of the ligand to the receptor. Finally, it was observed that the potentiating effects of IFN-gamma on LPS-induced G-CSF secretion could be blocked by interleukin-4 (IL-4). These data indicate that two cytokines produced by activated T cells have opposite effects on G-CSF production by human activated monocytes.

Interleukin-4 mRNA and protein in activated human T cells are enhanced by interleukin-7.

We studied the effect of the stroma-derived cytokine interleukin-7 (IL-7) on the expression of IL-4 in human T cells at mRNA and protein level. The results demonstrate that IL-7 did not induce IL-4 mRNA in resting T cells. However, concanavalin A (con A)-induced IL-4 mRNA expression was enhanced by costimulation with con A plus IL-7. Nuclear run-on analysis revealed that IL-7 did not affect the transcription rate of the IL-4 gene. The half-life of con A-induced IL-4 transcripts, however, was increased upon con A plus IL-7 treatment, indicating that the effect of IL-7 is mediated at posttranscriptional level. In accordance with the mRNA results, IL-4 protein was not detected in supernatants of unstimulated T cells or T cells exposed to IL-7. In contrast, IL-7 augmented the con A-induced secretion of IL-4 protein significantly. In addition, it was noticed that anti-IL-1 beta and anti-tumor necrosis factor-alpha (anti-TNF-alpha) did not abolish the effect of IL-7 on the con A-induced IL-4 secretion, indicating that the IL-7 effect is not mediated by the release of these cytokines. These results indicate that a stroma-derived factor can affect IL-4 expression in activated human T cells.

Recombinant human mast cell growth factor supports erythroid colony formation in polycythemia vera in the presence and absence of erythropoietin and serum.

The effect of mast cell growth factor (MGF) was studied on erythropoietin (Epo)-dependent and Epo-independent ("spontaneous") erythroid colony formation in patients with polycythemia vera (PV). MGF stimulated both Epo-dependent and Epo-independent erythroid colony formation from PV peripheral blood progenitor cells in vitro at a dose similar to normal erythroid progenitor. In addition, evidence was obtained that the stimulating effect of MGF was a direct effect on the erythroid progenitor and independent of serum. Antibodies against interleukin-1 (IL-1), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Epo could not abolish the enhancing effect of MGF. This was also supported by the finding that sorted CD34+ cells could be stimulated by MGF in the presence and absence of Epo. Finally, it was demonstrated that the spontaneous erythroid colony formation could not be ascribed to spontaneous release of MGF in the culture medium since anti-MGF did not affect the colony numbers. In conclusion, MGF has a direct stimulatory effect, independent of serum, on both Epo-dependent and Epo-independent erythroid colony formation in PV.

Erythroid progenitors in polycythemia vera demonstrate a different response pattern to IL-4 compared to the normal BFU-E from peripheral blood.

Human recombinant interleukin 4 (IL-4) was studied for its effects on erythroid burst-forming units (BFU-E) from normal peripheral blood and from patients with polycythemia vera (PV). IL-4 enhanced the proliferation of normal peripheral blood BFU-E (183% +/- 20% enhancement), whereas in the presence of interleukin 3 (IL-3) no further augmentation was noticed. The IL-4-mediated effects were independent of the absence or presence of adherent cells, B cells, or T cells. These data are in contrast with results obtained from normal human bone marrow cells, in which IL-4 antagonized the enhancing effects of IL-3. In PV a different response pattern was observed. The effects of IL-4 on the erythropoietin (Epo)-independent BFU-E were variable. In five PV patients no suppressive or enhancing effects of IL-4 were observed, whereas in two additional patients a significant decline in the Epo-independent BFU-E was noted. In the presence of IL-3, IL-4 significantly antagonized the IL-3-supported Epo-independent BFU-E in all patients (272% +/- 57% vs 187% +/- 49% enhancement, p less than 0.05). In contrast, IL-4 did not modify the IL-3-supported Epo-dependent BFU-E. In summary, these data suggest a difference between the normal and PV peripheral blood BFU-E. The Epo-dependent erythroid progenitors in PV patients showed a response pattern with IL-3 and IL-4 comparable to that of normal peripheral blood BFU-E, whereas the Epo-independent erythroid progenitors behaved like normal human bone marrow BFU-E, suggesting a shift in the stem cell compartment in PV. This is further supported by the finding that erythroid colony-forming units (CFU-E), normally only present in the bone marrow, could be cultured from the peripheral blood of PV patients in the presence or absence of Epo.

Aggressive chemotherapy for acute leukaemia frequently causes intestinal protein leakage.

Cytostatic drugs are known to produce disturbances in intestinal absorption of carbohydrates. To further explore the gastrointestinal (GI) toxicity of cytostatic therapy, 37 patients with acute leukemia were investigated during and/or after remission induction courses by the use of the differential sugar absorption test (DSAT) and the intestinal clearance of alpha-1-antitrypsin (ClAAT). The ratio of the lactulose to the mannitol excretion in the urine was found abnormal in 44% of the tests. The ClAAT was increased in 74% of tests. The tests results differed considerably from patient to patient and depended on the chemotherapy course; correlation between the tests was low, probably indicating the unrelated pathophysiological processes were measured. After haematological regeneration, abnormal test results normalised. It is concluded that aggressive chemotherapy not only causes a reduction in the absorption of sugars, but commonly also protein leakage. These GI side-effects are reversible, and the application of both tests in combination provides a practical and reproducible method for investigation of GI toxicity in patients treated with cytostatic drugs.

Susceptibility of the expression of parallel tubular structures in lymphocytes to the exposure to ammonium chloride buffer.

In isolation procedures for lymphocytes, isotonic ammonium chloride buffers are frequently used to lyse erythrocytes. In addition to evidence for an effect on lymphocyte function we now report a morphological alteration after treatment of lymphocytes with ammonium chloride buffer. Expression of parallel tubular structures was lost and only amorphous, electron-dense granules could be observed.

Hypothyroidism leads to more small-sized platelets in circulation.

The effect of induced hypothyroidism on platelet count and platelet volume distribution was studied in twelve athyreotic patients. After a two weeks withdrawal of triiodothyronine supplementation, platelet count and the ratio between platelet and red cell count were increased in all patients. Furthermore, mean platelet volume was declined and platelet distribution width was risen. Thus, hypothyroidism appears to increase the number of circulating platelets, especially the smaller ones.

The course of preexistent immune abnormalities in HIV negative haemophiliacs treated for two years with a monoclonal purified factor VIII concentrate.

The administration of factor VIII concentrates has been associated with immune abnormalities in patients with severe haemophilia A, even in the absence of HIV infection. The effects of a monoclonal purified factor VIII concentrate, Hemofil M (Baxter), on preexistent immune abnormalities were assessed over a 2 year period in 22 HIV negative haemophiliacs. They were treated previously with other concentrates, and received Hemofil T from 1983 to 1988. No HIV infection was demonstrated. No serologic evidence for other viral (re)-infections was seen. A decrease of HLA-DR expression on non-B lymphocytes in the first year (P = 0.026), and a decrease of T4-T8 ratio over the 2 years were found (P = 0.0016). Skin tests were non-contributive. The decrease in HLA-DR expression is suggestive for an improved immune function, possibly due to a reduced content of protein or virus in this concentrate.

Changes of buoyant density during the S-phase of the cell cycle. Direct evidence demonstrated in acute myeloid leukemia by flowcytometry.

Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows that the cellular buoyant density increases slightly with up to 0.008 g/ml during the S-phase, at least in cryo-preserved cells used in this study. This contrasts with the generally accepted belief that S-phase cells have a lower or constant buoyant density. A practical implication is that separation of cell (sub)populations based on differences in buoyant density could be flawed to the extent that these populations contain S-phase cells.

Recovery of murine myelopoiesis after cytostatic reduction by Ara-C. Effect of bacitracin-induced changes in the intestinal microflora and influence of timing.

The influence of intestinal flora modulation by oral bacitracin on the recovery of myelopoiesis after Ara-C was studied in C3H/Law mice. Bacitracin resulted in a 3-5 log increase of Gram-negative bacteria and a 10-fold increase of the intestinal endotoxin concentration. Initiation of bacitracin before Ara-C stimulated the initial rebound increase of colony-forming units for granulocytes and macrophages (CFU-GM) from 23.2 +/- 1.3 to 28.4 +/- 1.4 x 10(3) per femur. Starting the bacitracin after Ara-C advanced the second phase of the rebound CFU-GM increase with 6 days. An important role in the recovery of myelopoiesis after cytostatic drugs in C3H/Law mice is suggested for the intestinal Gram-negative microflora, probably mediated by bacterial endotoxin.

Influence of high versus low intestinal concentration of gram-negative bacteria and endotoxin on the susceptibility of murine myelopoiesis in bone marrow and spleen to cytostatic treatment with Ara-C.

The haematopoietic recovery after i.v. cytarabine was studied in C3H/Law mice as a measure for stem cell susceptibility in relation to the intestinal Gram-negative bacteria (GNB) and endotoxin. Reduction or elevation of GNB and endotoxin was induced by either polymyxin or bacitracin, both non-absorbable antibiotics. Bacitracin caused less suppression of the splenic cellularity after cytarabine, and an advancement of the recovery of femoral nucleated cells. The femoral recovery of CFU-GM exhibited a biphasic pattern. The speed and height of the rebound increase of CFU-GMs were significantly affected by the antibiotics. Thus, (modulation of) the murine intestinal microflora influences the haematopoietic recovery after cytostatic drugs. The mechanisms involved are complex; intestinal endotoxin seems to play a role.

Pseudothrombocytopenia due to agglutinins.

Pseudothrombocytopenia may have any of a number of causes, one of which is agglutination in vitro. This phenomenon was found in samples of blood from six patients. A serum factor responsible for the agglutination was demonstrated. The factor was dependent upon the presence of EDTA and was more active at room temperature than at 37 C. It could be identified as an IgM immunoglobulin in four cases. In the other two cases definite characterization was not possible, but there was some evidence in favor of an IgM factor. All six patients had elevated serum IgM levels, but they had different and unrelated clinical disorders.

Plasma spermidine concentrations as early indication of response to therapy in human myeloma.

Eighteen patients with melphalan refractory myeloma were treated with vindesine and prednisone. Plasma spermidine concentrations were measured by radioimmunoassay before and after a single vindesine injection. Seven patients showed a significant rise of plasma spermidine after vindesine and five of these showed a clinical response on further evaluation. Of the 11 patients who did not show raised spermidine concentrations, 10 did not respond to the therapy. The correlation between clinical response/rise of spermidine and between non-response/no rise of spermidine was statistically significant (p less than 0.05). Pretreatment spermidine concentrations did not distinguish those who responded to treatment nor did they differ in patients and controls.

Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase.

A discrepancy exists between basal tissue factor (TF) expression found in endothelial cell cultures and the failure to detect TF in unpertubated endothelial cells in vivo. We demonstrated that basal TF expression in endothelial cell cultures originated from contaminating cells. These cells were ultrastructurally and flowcytometrically identified as smooth muscle cells. The cell cultures had been obtained from collagenase-treated human umbilical cord vessels. Histologic studies revealed that after collagenase treatment the basement membrane was digested and underlying structures were disrupted at some areas of the vein. We selected chymotrypsin as an alternative for the isolation of endothelial cells. Using chymotrypsin, the endothelial lining was selectively lost leaving the basement membrane undisturbed. Furthermore, use of chymotrypsin instead of collagenase minimized the level of basal TF activity. Taken together, we demonstrated that basal TF expression in endothelial cell cultures is caused by contaminating smooth muscle cells. This contamination can strongly be reduced using chymotrypsin instead of collagenase for isolation of endothelial cells.

Urinary polyamines and their metabolites during new combination chemotherapeutic treatments of high grade non-Hodgkin lymphoma.

Nineteen patients with non-Hodgkin lymphoma of unfavourable histology (15 high grade and 4 intermediate grade) were treated with two new combination chemotherapeutic schemes. Except for one all were partial (8) or complete (10) responders to treatment. Polyamines were measured in every spontaneously voided urine sample. Pretherapeutically all (11) stage III and IV patients had borderline or increased urinary putrescine (Pu) and sum of isoputreanine, spermidine and spermine (sigma Isoputr,Sd,Sp), except for the non-responder. Except for one, all (8) stage I and II patients had normal urinary Pu and sigma Isoputr,Sd,Sp. Posttherapeutically patients with pretherapeutically increased sigma Isoputr,Sd,Sp returned to normal (5), borderline (2), or slightly increased (3) levels. The post-therapeutic achievement of normal or borderline sigma Isoputr,Sd,Sp was not necessarily connected with accomplishment of complete remission. From the start of therapy until clinical restaging, partially or completely responding stage III and IV patients excreted 5-234 mmol sigma Isoputr,Sd,Sp per mol of creatinine above the mean normal value plus 2 SD. For stage I and II patients and the clinical non-responder this parameter amounted to 0-5 mmol/mol of creatinine. Peaks in urinary Pu and sigma Isoputr,Sd,Sp follow-up curves were related in time to the administration of chemotherapeutics. For responding stage III and IV patients the rate of the decrease of sigma Isoputr,Sd,Sp levels paralleled the clinically observed rate of tumour load reduction. This study suggests that notably for non-Hodgkin lymphoma patients with high tumour loads the constant monitoring of polyamines can provide information on pretherapeutic spontaneous tumour cell loss, the efficacy of chemotherapeutic combinations, the kinetics-, and (within certain limitations) the extent of therapeutically induced tumour cell death.