Granulocytic Subtypes Research Articles

Electrophysiological properties of three types of granulocytes in circulating blood of the newt.

1. The electrophysiological properties of three subtypes of granulocytes obtained from the circulating blood of the newt, Triturus pyrrhogaster, were studied using the whole-cell variation of the patch-electrode voltage-clamp technique. 2. Neutrophils were identified by their multilobulated nucleus in the cytoplasm. Basophils and eosinophils, having characteristic granular structures in their cells, could definitely be distinguished from other leucocytes. The reliability of cellular identification using phase-contrast microscopy was confirmed by fixing and staining the granulocytes with Wright's solution. 3. In neutrophils under current-clamp conditions, a hyperpolarization-induced conductance increase was observed. With depolarization, however, no changes in regenerative potential were detected. When voltage clamped in standard saline (containing 96 mM-NaCl), hyperpolarizing voltage pulses to a potential more negative than -90 mV evoked slowly decaying inward currents. 4. The hyperpolarization-evoked membrane currents in neutrophils were identified as anomalous rectifying K+ currents, since (1) externally applied Cs+ (0.1 or 1 mM) or Ba2+ (1 mM) produced suppressive effects on the currents, (2) replacement of external Na+ with choline ions eliminated the decay of macroscopically observed currents, and (3) both the amplitude and kinetic properties of the currents were strongly dependent on membrane potential as well as on external K+ concentration; the activation of the conductance depended on the electrochemical force for K+ rather than on membrane potential alone. The magnitude of steady-state conductance was roughly proportional to the square root of the external K+ concentration. 5. In basophils and eosinophils, no major time- or voltage-dependent increase in conductance was detected at voltages between +20 and -130 mV. However, under current-clamp conditions, spontaneous fluctuation of zero-current potentials was clearly apparent, presumably due to the activities of some ion channels generating a small amount of current flux through the membranes of these cells. 6. It was concluded that the three subtypes of granulocytes in the newt differ considerably not only in appearance and structure but also in the electrical properties of their membranes. The anomalous rectifying K+ channels in neutrophils may serve to determine the resting potential of the cell at K+ equilibrium potential. The closure of the channels at depolarization might facilitate the maintenance of depolarization triggered by stimuli accompanying current influx.

Pronephric leucocytes of Cyprinus carpio: Isolation, separation and characterization

Since the teleost pronephros is an important source of diverse immunocytes, suspensions of pronephric cells from young adult carp have been characterized. In freshly prepared suspensions, adherent, spreading cells (macrophages?) constituted <3% of the total population. Granulocytes and lymphocytes were co-dominant (<80%) leucocyte types. Continuous Percoll density gradient centrifugation yielded discrete subpopulations with these ϱ values and cytological characteristics: Fraction I & II ϱ = 1.055 − 1.070 thrombocytes, monocytes, macrophages, and lymphocytes. Fraction III ϱ = 1.080 − 1.090 granulocytes, type 1. Fraction IV ϱ = 1.105 − 1.110 erythrocytes and granulocytes, type 2. Fraction V ϱ = 1.118 − 1.125 granulocytes, type 3. Fraction VI ϱ = 1.140 − 1.150 granulocytes, type 4. Granulocyte motility increased markedly over the first 24 hr in vitro, and was enhanced by components washed from intact yeast. The subtypes of granulocytes were distinguishable by not only the ϱ values, but also on the basis of cell size, ultra-structure of the granules, and their histochemical and phagocytic characteristics. After simultaneous in vivo injection of Bacillus megaterium (Gram + ve), Aeromonas hydrophila(Gram − ve) and Saccharomyces cerevisiae (yeast), individual pronephric leucocytes were found capable of phagocytosing all three types of particle. Granulocytes which had phagocytosed B. megaterium were slower than macrophages in their ability to kill the bacteria. Encounter with B. megaterium or S. cerevisiae in vitro elicited a clumping reaction which involved mostly the larger leucocytes [granulocytes]. Both adherent cells and non-adherent cells were phagocytic in vitro.

Surface markers on leukemia and lymphoma cells: recent advances

Recent advances in immunology have led to important insights into leukocyte differentiation and the cellular origin of leukemia. It is now possible to precisely define stages of human lymphocyte and granulocyte differentiation utilizing highly specific monoclonal antibodies that define cell surface antigens in conjunction with more traditional markers such as surface and cytoplasmic immunoglobulin on B lymphocytes, sheep erythrocyte (E) receptors on T lymphocytes, and cytochemical staining of myeloid cells. Five major subtypes of acute lymphoblastic leukemia (ALL) are now recognized, including unclassified or null ALL, common ALL (cALL), pre-B-ALL, B-ALL, and T-ALL. Within the T-ALL subgroup, there is considerable heterogeneity based on the presence or absence of the E-receptor and various T-cell surface membrane antigens defined by monoclonal antibodies. Cells from patients with chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and Sέzary syndrome also can be classified by their reactivity with monoclonal antibodies. Acute myelogenous leukemia (AMD cells are generally classified by morphological and cytochemical features. Recently, monoclonal antibodies reactive with granulocytic and monocytic subtypes of AML have been developed. In addition to their utility in leukemia classification, monoclonal antibodies that identify leukemia associated antigens have also been used therapeutically. Monoclonal antibodies have been infused intravenously into patients with leukemia and lymphoma. They have also been used in vitro with complement to lyse residual leukemia cells from remission bone marrows removed from leukemia patients. These treated bone marrows were later reinfused into the same patients to “rescue” them following high-dose chemotherapy and radiation therapy.

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.