calcimycin and 3-3--dipentyl-2-2--oxacarbocyanine

Defective phagocytic cell function may partially account for the morbidity and mortality associated with thermal injury. In experimental thermal injury in the rat, small circulating blood volumes increase the difficulty in obtaining significant data. Furthermore, purification and or elicitation procedures have the potential for altering the cell surface characteristics and/or the functional response of the cell in question. We have examined the circulating neutrophils and pulmonary alveolar macrophages of anesthetized rats following a 16-20% body surface area scald injury to the shaved back. The circulating neutrophils of thermally injured rats were examined by flow cytometry following stimulation with phorbol myristate acetate (PMA) (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric cyanine dye, dipentyloxocarboxyanine and the formation of the oxidized product of 2',7'-dichlorofluorescin diacetate-loaded cells. The alveolar macrophages were examined after stimulation with PMA (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric dye, dipropylthiodicarbocyanine and the generation of superoxide production, as assessed by the superoxide dismutase inhibitable reduction of cytochrome c. Both cells exhibited a profound inhibition of cell function 4 hours after the insult, with partial return toward control values at later time points. Furthermore, the plasma of thermally injured rats, 4 hours after the burn was inhibitory to normal rat neutrophils. Fluorescent compounds suggestive of in vivo lipid peroxidation were maximally detectable at this time point. Further research is needed to establish the role of these products in the induction of phagocytic cell dysfunction.

Topics: Animals; Burns; Calcimycin; Carbocyanines; Flow Cytometry; Fluoresceins; In Vitro Techniques; Leukocyte Count; Macrophages; Neutrophils; Oxygen; Phagocytes; Plasma; Rats; Rats, Inbred Strains; Superoxides; Tetradecanoylphorbol Acetate

Stimulated neutrophils show ionic fluxes that may function as "transducers" between stimuli and effector functions. Using fluorescent probes, patterns of membrane-associated calcium (chlortetracycline, CTC) and membrane potential (3-3'-dipentyloxacarbocyanine, di-O-C5 (3)) in single living human neutrophils were observed with a fluorescence microscope fitted with an image intensifier and photometer. Fluorescence changes were related to chemiluminescence. In unstimulated neutrophils, CTC and di-O-C5 (3) fluorescence was brightest in the perinuclear region. Di-O-C5 (3) fluorescence was also seen in mitochondria. Neutrophil stimulation with zymosan, phorbol myristate acetate (PMA) or calcium ionophore (A23187) resulted in loss of di-O-C5 (3) and CTC fluorescence and chemiluminescence proportional to the strength of the stimulus. Experiments demonstrated the independence of these processes. (1) Digitonin stimulation caused chemiluminescence and di-O-C5 (3) darkening without loss of CTC fluorescence. (2) Depolarization of neutrophils did not induce CTC darkening or chemiluminescence. (3) Calcium ionophore (A23187) stimulation of neutrophils in calcium-free medium resulted in normal di-O-C5 (3) and CTC darkening, but a blunted chemiluminescence peak. (4) Calcium ionophore (A23187) stimulated the loss of di-O-C5 (3) and CTC fluorescence from chronic granulomatous disease neutrophils, but did not trigger an oxidative burst. Although neutrophil depolarization, calcium release from membranes, and oxidative activity are linked, these processes can clearly be separated.

Topics: Animals; Calcimycin; Calcium; Carbocyanines; Cell Membrane; Chickens; Chlortetracycline; Digitonin; Egtazic Acid; Humans; Luminescent Measurements; Membrane Potentials; Microscopy, Fluorescence; Neutrophils; Oxytetracycline; Zymosan

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin