Response of human multiple myeloma-derived cell lines to alkyl-lysophospholipid.

Clinical studies using high-dose chemotherapy and autologous bone marrow transplantation in multiple myeloma report improvement in tumor regression. The potential success of this approach may be compromised because of the myeloma cell burden. A promising approach is chemopurging of bone marrow with alkyl-lysophospholipids (ALP), a new class of antitumor agents. Using a limiting dilution assay, we tested ALP against several drug-resistant human myeloma cell lines: RPMI 8226/S, 8226/DOX40, 8226/LR-5, 8226/MR-20, 8226/DOX1V, and 8226/MDR10V. IC50 values were approximately 6 microg/mL, for all cell lines evaluated. ALP was effective against all cells, with mean log kills ranging from 2.48 to 4.95. Cytotoxicity was temperature-dependent. Survival curves of cell lines exposed to ALP showed a steep slope during the first 4 hours of exposure, with little additional cell kill after 24 hours. Dose-response curves after drug exposure for 4 and 24 hours reached plateaus at 75 and 25 microg/mL, respectively. Increasing concentrations of ALP caused a sustained increase in resting [Ca2+]i of sufficient magnitude to induce cell death. These results suggest that ALP is an effective cytotoxic agent even in cells known to be resistant to alkylating agents and other classes of cytotoxic drugs.

Functional characteristics of in vivo induced neutrophils after differentiation therapy of acute promyelocytic leukemia with all-trans-retinoic acid.

The authors describe the functional capabilities of in vivo induced neutrophils from a patient with acute promyelocytic leukemia (French-American-British M3v) treated with differentiation therapy using all-trans-retinoic acid (45 mg.m-2.day-1). The induced neutrophils from the leukemic clone appeared in the blood 7 days after therapy. Normal neutrophils, presumably derived from nonclonal normal hematopoiesis, appeared 15 days after the initiation of therapy. The induced neutrophils were separated from normal neutrophils by density gradient centrifugation. Their origin was established by fluorescence in situ hybridization. The induced neutrophils were morphologically atypical but stained for myeloperoxidase (Sudan black B) and AS-D chloroacetate esterase and were negative for alpha-naphthyl butyrate esterase. Induced neutrophils were functionally mature, showing nitroblue tetrazolium reduction in 72% of the cells compared with 84% in the normal neutrophil fraction. Both the rate and total killing of Staphylococcus aureus (American Type Culture Collection Strain 25923) were normal in both neutrophil fractions. Random locomotion was equivalent and within the normal reference range in both fractions; however, using the under-agarose technique, induced neutrophils showed a minor chemotactic defect in response to both n-formyl-methionyl-leucyl-phenylalanine (score 292, normal 338-868) and complement-derived chemotactic factors (score 420, normal 457-1408). At autopsy, induced neutrophils infiltrated necrotic myocardial tissue, suggesting a normal response to inflammatory stimuli.

The effect of various cell separation procedures on assays of neutrophil function. A critical appraisal.

Neutrophils are considered fragile cells, easily damaged by improper handling. Assays of neutrophil function frequently require preliminary isolation procedures that may be potentially harmful. The authors did an extensive investigation of the effects of currently used isolation procedures on a broad spectrum of functional assays that included: 1) phagocytosis; 2) bacterial killing by a culture technique and tritiated thymidine labeling; 3) candidacidal activity by methylene blue dye exclusion and differential Giemsa staining; 4) quantitative unstimulated and histochemical stimulated nitroblue tetrazolium dye reduction; 5) chemiluminescence; 6) random locomotion; and 7) chemotaxis by the agarose method. In addition, cells were examined morphologically by electron microscopy, and the granule density was quantitated by morphometric analysis. Isolation techniques included erythrocyte sedimentation, hypotonic lysis, gradient density separation using Ficoll-Hypaque, and counterflow centrifugal elutriation. The purest neutrophil suspensions were obtained by density gradient separation and counterflow centrifugal elutriation with mean neutrophil percentages of greater than or equal to 94%. Regardless of the isolation procedure, neutrophils were similar in all groups (P greater than .05) in the following studies: phagocytosis, nitroblue tetrazolium dye reduction, bacterial killing, candidal killing, inhibition of candidal germ tube formation, and cytoplasmic granulation. Differences were noted in assays of neutrophil migration and chemiluminescence. Neutrophil suspensions isolated by counterflow centrifugal elutriation and Ficoll-Hypaque had the highest scores for random migration and chemotaxis. These differences can be related to the purity of the neutrophil suspension rather than the harmful effects of the isolation procedures. Erythrocyte contamination affected both the slope and the time to peak response in the chemiluminescence assay. Exposure of neutrophils to cold temperatures (0-4 degrees C) for 1.5 hours impaired both random locomotion and chemotaxis. Current recommendations for the storage, transport, and preparation of leukocytes for neutrophil function studies need to be reassessed.

Functional differentiation of normal human neutrophils.

In the past differentiation of human neutrophils has been defined by morphology, cytochemistry, or surface markers. In our experiments we have sequenced the various events that occur during the functional differentiation of the normal human neutrophil and have also examined some of the functional properties in relationship to surface markers and biochemical events. Granulocytes were obtained from the bone marrow and blood of hematologically normal individuals. Cells were separated into different stages of maturation by their physical properties using counterflow centrifugal elutriation and density gradient separation. Three cell fractions were obtained that were enriched for either immature myeloid cells, band neutrophils, or segmented neutrophils. Since the enriched fractions were not entirely pure, methodologies for functional assays were chosen that allowed cytologic evaluation of the functional capacity of each cell type. The criteria used to classify the stages of differentiation included both morphology by light microscopy and DNA labeling with tritiated thymidine. Various neutrophilic properties were studied: Fc receptors, complement receptors (CR1, CR3), phagocytosis of both live and dead opsonized Staphylococcus aureus, microbial killing of S aureus, NBT dye reduction after cellular stimulation with endotoxin, and chemotaxis. Our results indicate that the functional properties of the neutrophil appear in a distinct order. The sequence for the functional differentiation of the human neutrophil appears to be the following: Fc receptors----immune phagocytosis----complement receptors----oxygen-independent microbial killing----oxygen-dependent microbial killing----chemotaxis.

Bordetella pertussis adenylate cyclase: effects of affinity-purified adenylate cyclase on human polymorphonuclear leukocyte functions.

Affinity-purified adenylate cyclase (AC) of Bordetella pertussis, free of contaminating pertussis toxin, was demonstrated to have biological effects on human polymorphonuclear leukocytes (PMN). AC at doses of 25 and 50 micrograms/ml increased intracellular cAMP levels in the phagocytes 7.6- to 23.5-fold, respectively, above basal levels. AC inhibited PMN chemiluminescence, chemotaxis, and superoxide production in a dose-dependent manner. The 50% inhibitory dose for chemotaxis and chemiluminescence was 36.5 micrograms/ml; for superoxide generation it was 71.0 micrograms/ml. Although these PMN metabolic functions were impaired, no effect on phagocytic activity was observed.

Liquid preservation of human neutrophils stored in synthetic media at 22 degrees C: controlled observations on storage variables.

The purpose of this study was to use a chemically defined medium to identify essential substances and optimal conditions for the liquid storage of neutrophils at 22 degrees C. Several commercially available synthetic media were evaluated: L-15, McCoy's 5a, M199, minimum essential medium, Dulbecco's MEM, NCTC135, and RPMI 1640. Proteins, glucose, pH, and neutrophil concentration were systematically studied. Neutrophils were harvested by centrifugal cell separators or phlebotomy, and their maintenance was evaluated by monitoring cell counts, dye exclusion, phagocytosis, bacterial killing, and chemotaxis. Neutrophils stored equally well in all synthetic media except L-15; however, chemotaxis was poorly maintained in synthetic media as compared with autologous plasma. RPMI 1640 was arbitrarily selected as a basal medium to evaluate storage variables. RPMI 1640 supplemented with albumin to a concentration of 1% improved chemotaxis and was equivalent to plasma as a storage medium with regard to the in vitro functions tested. Cohn fractions IV-1, IV-4, and gamma globulin were not effective substitutes for albumin. Glucose is essential for neutrophil storage; its absence from the medium correlated with poor cell function. Optimal glucose requirements depend on the cell concentration. High glucose concentrations were toxic to neutrophils; at 1,000 mg/dL, chemotaxis was depressed by 58%. Glucose utilization was dependent on the initial pH of the medium and on the cell concentration. A wide range of hydrogen ion concentrations was tolerated, and the optimum pH range was 7.2 to 7.8. Cell concentration is an important variable because it affects the pH of the medium as well as glucose utilization.

Granulocyte concentrates. Glucose concentrations and glucose utilization during storage at 22 degrees C.

Neutrophil metabolism is dependent on the available stores of either exogenous or endogenous glucose. We studied 38 granulocyte concentrates collected by four different centrifugal cytapheresis systems: Haemonetics model 30, IBM 2997, Fenwal CS-3000, and Fenwal Celltrifuge II. Extracellular glucose concentrations were measured immediately following collection and after 24 hours of storage. The Fenwal CS-3000 produced the highest concentrations of platelets and leukocytes, and the units had the lowest initial glucose levels and pH. After 24 hours of storage, there was a mean glucose of 1.7 mg per dl in the Fenwal CS-3000 granulocyte concentrates and 9 of 12 units had no detectable glucose. In contrast, concentrates collected from other systems had sufficient glucose available to support neutrophil metabolism for a longer period.

The effect of platelets and red cells on granulocytes stored at 22 degrees C.

Granulocyte concentrates contain varying numbers of platelets and red cells depending upon the method of collection. Either platelet or red cell concentrations may be as high as 2.0 X 10(12) per I. Studies were done on unwashed and washed granulocyte concentrates and on pure granulocyte suspensions with known numbers of platelets or red cells added. These suspensions or concentrates were stored for 72 hours at 22 degrees C. In both experiments, the following were measured: leukocyte and absolute granulocyte counts, dye exclusion, chemotaxis, plasma glucose, plasma pH, and osmolality. Red cell contamination did not adversely effect granulocyte storage. Platelets, however, did contribute to the functional deterioration of stored granulocytes. In the presence of high concentrations of platelets, both granulocyte dye exclusion and chemotaxis were adversely affected at 48 hours of storage. In another experiment, fresh autologous granulocytes incubated for 18 hours in hydroxyethyl starch-citrated-plasma obtained from stored granulocyte concentrates showed a progressive decrease in chemotaxis related to the age of the stored plasma. Glucose supplementation of the spent plasma maintained chemotactic activity. Contamination of granulocyte concentrates with other cells and the availability of glucose to granulocytes are variables affecting the short-term liquid storage of granulocytes at 22 degrees C.

Neutrophil migration under agarose: quantitation and variables.

Several variables have been evaluated for the in-vitro measurement of neutrophil migration under agarose. Commonly used anticoagulants do not alter migration. Cells may be collected in heparin, sodium citrate, or EDTA. Neither hypotonic or ammonium chloride lysis of erythrocytes affects chemotactic activity. Saponin, however, decreased both leukocytic viability and migration. Passage of neutrophils through a Hypaque-Ficoll density gradient improved migration scores, presumably by removing contaminating material and cells. Both lymphocyte and erythrocyte contamination decreased test scores. Platelets did not have a detrimental effect on neutrophil migration. Fresh serum was the most potent chemotactic agent. This was followed, in order, by zymosan-activated serum, N-formyl-L-methionyl-L-phenylalanine, casein, and Escherichia coli supernatant. A scoring method that combines features of both counting leukocytes and measuring the migration distance has been devised. The technic has a coefficient of variation of 9.2%. Scores for the normal adult population show a gaussian distribution.