Action of organophosphorus compounds on the p-nitrophenyl phosphatase of membranes from rabbit and guinea pig polymorphonuclear leucocytes

Organophosphorus compounds such as DFP and its non-phosphorylating analogue, triisopropyl phosphate, stimulate the p-nitrophenyl phosphatase activity of membranes from rabbit and guinea pig leucocytes. Triethyl phosphate and trimethyl phosphate have little effect and the stimulation by triisopropyl phosphate can be distinguished from that of the non-ionic detergent lubrol. It is suggested that the effect on the membrane leading to p-nitrophenyl phosphatase stimulation can be the basis of the inhibition of locomotion and the enhancement of leucocidin action in the leucocyte. The hypothesis that Organophosphorus compounds act on cells exclusively as inhibitors of esterases is criticised. The p-nitrophenyl phosphatases of rabbit and guinea pig leucocyte membranes differ in some respects and it is suggested that a study of the enzymes in related cells may reveal further species differences.     

Notes on Technic: Differential staining of Fungus and host cells Using a modifciation of Pianeze IIIB

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Cyto-immunological studies of guinea pig sperm antigens

Summary Antigenic localization in guinea pig epididymal sperm and testicular imprints as well as in viable, motile guinea pig epididymal sperm was studied by means of fluorescent labelled antibody techniques. Globulins from rabbits and chickens immunized with guinea pig epididymal sperm were used in the direct procedure while sera from sheep and fowl injected with rabbit globulins were used in the indirect procedure. The main findings were: 1) spermatozoa from the distal portion of the epididymis displayed brilliant fluorescent acrosomes and less intensely stained midpieces and principal pieces when treated as dried smears in both the direct and indirect methods; 2) testicular spermatozoa were similarly stained but whereas in epididymal spermatozoa the whole acrosome stained intensely, the testicular spermatozoal acrosome displayed intense fluorescence of the inner acrosome; 3) protoplasmic droplets fluoresced strongly; 4) cross-reactivity was observed between human and guinea pig sperm but not between rat and guinea pig sperm, indicating an antigenic relationship between human and guinea pig but not between guinea pig and rat; 5) treatment of viable, motile guinea pig spermatozoa with fluorescent globulins resulted in agglutination and immobilization as well as formation of antigen-antibody aggregates adherent to the cell membrane of the head, midpiece and principal piece; the formation of such fluorescent aggregates in the medium surrounding the treated motile sperm was indicative of leaching of antigenic material from the sperm cells.This investigation was supported by funds from United States Public Health Service grant HE-05798-03, The Ford Foundation and National Science Foundation.     

Staining Secretory Capillaries of Exocrine Glands with Techniques for Specific Phosphatases

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Partial purification, physicochemical characterization and biological function of a rosette-decreasing factor (RDF) extracted from guinea pig thymus.

A factor that decreases rosette formation between guinea pig T-cells and rabbit red blood cells (RRBC) was extracted from the thymus of the guinea pig. The active factor could be extracted from the spleen as well as the thymus, but not from the liver or kidney. The active factor of the thymic extract was found in the precipitates produced by 80% saturated ammonium sulfate and it was separated from the water-soluble fraction of the precipitates. The molecular weight of the partially purified substance was estimated to range between 10,000 and 30,000 by filtration through a diaflow membrane. From the studies on physicochemical characterization, it might be a heat-resistant basic peptide probably bound to a ribonucleotide moiety. This factor reduced rosette formation between RRBC and guinea pig T-cells, but did not reduce erythrocyte-antibody-complement rosette formation. This factor also inhibited mitogen (concanavalin A, phytohemagglutinin-P)- induced DNA synthesis of guinea pig lymphocytes and antigen-induced DNA synthesis of sensitized guinea pig lymphocytes.     

The inhibition of locomotion of the polymorphonuclear leucocyte by organophosphorus compounds

The effects of diisopropylphosphorofluoridate (DFP) and other organophosphorus compounds on the locomotion of rabbit polymorphonuclear leucocytes have been investigated in vitro using time-lapse cinémicrography. Both phosphorylating and non-phosphorylating compounds were observed to inhibit cell locomotion, not only increasing the proportion of stationary cells, but also decreasing the velocity of those cells whose movement continued. This inhibition of locomotion occurred over the same concentration range of organophosphorus compound which was previously found to enhance the effect of leucocidin on the leucocyte. Although the inhibitory effects of low concentrations of organophosphorus compounds were partly reversible, higher concentrations produced effects which continued to increase even after the cells had been returned to normal medium. It is suggested that the supposed effect of organophosphorus compounds on chemotaxis may actually be due to the inhibition of locomotion per se, probably through the detergent properties of these compounds rather than their properties as enzyme inhibitors.     

Guinea pig macrophage agglutination factor is antigenically distinct from migration inhibition factor and immunoglobulin.

Lymph node cells from guinea pigs with specific delayed hypersensitivity release macrophage agglutination (MAggF) and migration inhibition factors (MIF) upon exposure to antigen or concanavalin A in serum-free medium. MAggF in culture supernatants was absorbed neither by immunoabsorbents made with a rabbit anti-guinea pig lymphokine serum that removed MIF, nor by immunoabsorbents made with rabbit anti-guinea pig Ig. These results suggest that MAggF is antigenically distinct from MIF and Ig.     

Studies on thymocyte subpopulations in guinea pigs. VIII. Characterization of a thymocyte population resistant to the cytotoxic effect of normal rabbit serum

Guinea pig thymocytes were incubated with normal rabbit serum, which resulted in the death of a great majority of the cells. The remaining rabbit serum-resistant cells, representing less than 10% of the thymocytes, contained euchromatic DNA and were of intermediate size and low density. Functional tests indicated that they were enriched in immunologically mature cells, which responded to the mitogenic lectins phytohemagglutinin and concanavalin A, and were depleted of immature, spontaneously proliferating cells and in cells responding to the thymocyte growth peptide. The described procedure for enrichment of immunologically mature thymus cells in guinea pigs may become useful since glucocorticoid treatment, used in mice for enrichment of mature thymocytes, cannot be used for this purpose in guinea pigs.     

Secretion of intrinsic factor from dispersed mucosal cells isolated from guinea pig and rabbit stomach

In dispersed mucosal cells prepared from rabbit and guinea pig stomach, the secretion of intrinsic factor was constant (0.3–0.4%/min) for at least 30 min incubation at 37°C. Histamine or isobutyl methylxanthine increased cyclic AMP and intrinsic factor secretion in both cell preparations. Isobutyl methylxanthine potentiated and cimetidine competitively inhibited (K_i=5·10^?7 M) both effects of histamine. Dibutyryl cyclic AMP (1.0 mM), also caused a 3-fold increase in intrinsic factor secretion. These results suggest that in rabbit and guinea pig histamine interacts with H₂-receptors to increase cyclic AMP which mediates the rise in the rate of intrinsic factor secretion.     

FACTORS AFFECTING THE REDISTRIBUTION OF SURFACE-BOUND CONCANAVALIN A ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES

Human neutrophil polymorphonuclear leukocytes (PMN) were studied to determine the influence of cellular locomotion upon the redistribution and capping of concanavalin A (Con A). Con A was detected by fluorescence (using Con A conjugated to fluorescein isothiocyanate [Con A-FITC]), or on shadow-cast replicas (using Busycon canaliculatum hemocyanin as a marker for Con A). After labeling with Con A 100 µg/ml at 4°C and warming to 37°C, locomotion occurred, and the Con A quickly aggregated into a cap at the trailing end of the cell. When locomotion was inhibited (with cytochalasin B, or by incubation in serum-free medium at 18°C) Con A rapidly formed a cap over the central region of the cell. Iodoacetamide inhibited capping. PMN labeled with FITC, a monovalent ligand, developed caps at the tail only on motile cells; FITC remained dispersed on immobilized cells. PMN exposed to Con A 100 µg/ml at 37°C bound more lectin than at 4°C, became immobilized, and showed slow central capping. The Con A soon became internalized to form a perinuclear ring. Such treatment in the presence of cytochalasin B resulted in the quick formation of persistent central caps. Colchicine (or prior cooling) protected PMN from the immobilizing effect of Con A, and tail caps were found on 30–40% of cells. Immobilization of colchicine-treated cells caused Con A to remain in dispersed clusters. Thus, capping on PMN is a temperature- and energy-dependent process that proceeds independently of cellular locomotion, provided a colchicine-sensitive system is intact and the ligand is capable of cross linking receptors. On the other hand, if the cell does move, it appears that ligands may be swept into a cap at the tail whether cross-linking occurs or not.     

Kinetic evidence for a common mechanism of capping on lymphocytes

1. Differences in the rates at which ligands cap various receptors on the same cells, and their sensitivity to various drugs, have been interpreted as evidence that there are distinct mechanisms for `fast' and `slow' cap formation. We have examined the factors which determine the rate of cap formation of three receptors on mouse splenic lymphocytes or thymocytes, and compared the effects of cytochalasin B or colchicine under conditions where the different receptors cap at similar rates. 2. When surface immunoglobulin, concanavalin A receptors, or θ antigen are induced to cap at their maximal rates by appropriate concentrations of one or more cross-linking ligands, the half-time for maximal capping of each receptor population is between 1.5 and 3.0min at 37°C. Slower rates of cap formation are obtained by using non-optimal concentrations of the cross-linking ligands. 3. When the three receptors were induced to cap at similar rates (either maximal or slower), 10μm-cytochalasin B caused a similar decrease in the rate of cap formation for each receptor, without affecting the eventual extent of capping. At comparable capping rates on control cells, colchicine (10μm) increased the rate of cap formation for surface immunoglobulin and concanavalin A receptors to a similar extent, without affecting the eventual extent of cap formation. In contrast, colchicine had no detectable effect on the capping of θ antigen. 4. From these results, we conclude that there are no intrinsic differences in the rates at which different receptors can be induced to cap that can be used to diagnose differences in their mechanisms of cap formation. The observation that ligand concentration and the drugs acting on the cytoskeleton generally affect the rate but not the extent of cap formation accounts for the wide variation in reported effects of the drugs on cap formation measured at fixed times. The receptor-specific effect of colchicine on surface immunoglobulin and concanavalin A receptors, but not θ antigen, is not readily compatible with models of cap formation which depend on lipid or membrane flow.     

Action of cytochalasin E on polymorphonuclear leucocytes of guinea pig peritoneal exudates

Actions of cytochalasins on polymorphonuclear leucocytes of guinea pig peritoneal exudates have been studied. When the leucocytes were treated with cytochalasin E, complete disappearance of pseudopods was observed during a time, which would be related with the inhibition of particle ingestion. Intracellular granules migrated to the cell periphery in ectoplasma and were afterwards excluded from the cells. Later, formation of cytoplasmic protuberations with irregular shapes and variable diameters (“zeiosis”) and of large vacuoles were observed. Similar changes were also observed in monocytes. These morphological changes were accompanied with metabolic alterations which mimicked those observed during phagocytosis: appearance of cyanide-insensitive respiration, stimulation of hexose monophosphate oxidative pathway and release of superoxide anions and lysosomal enzymes into suspending medium. Other members of the cytochalasin family, cytochalasin C and D, similarly induced release of superoxide anions, whereas cytochalasin A and B had no such effect on leucocytes. Cytochalasin E seemed to have dual effects on leucocytes, namely, (1) inhibition of cytokinesis which is a common effect of the cytochalasin family; and (2) triggering of metabolic changes and degranulation which are characteristics of phagocytotic process.     

Comparative histochemical observations on the contributions of the acrosomal vesicle and granule to the acrosomal cap in the spermatozoa of mammals

Summary By applying the periodic acid-Schiff technique to frozen sections of material fixed in weak Bouin's solution and extracted with hot pyridine, the contributions of the acrosomal vesicle and granule to the acrosomal cap have been observed in the maturing spermatozoa of the American opossum, guinea pig, rabbit, marmoset and chimpanzee. In the spermatids of the opossum, the acrosomal granule is not developed and the thin, homogeneous carbohydrate layer of the acrosomal cap is derived from the concentrated material of the acrosomal vacuole. In the guinea pig, both the acrosomal vesicle and granule make the greatest contributions to the carbohydrates of the acrosomal cap; these continue to maintain their identity in the head of mature sperm. In the rabbit, marmoset and chimpanzee, the carbohydrate material of acrosomal vesicle origin is less developed and constitutes a thin layer in the lateral portions of the acrosomal cap; the carbohydrate material in its anterior portions is mainly derived from the acrosomal granule which becomes flattened; there is also a slight thickening of the intermediate layer of the acrosomal cap in this region. The distinction between the carbohydrate contributions from the acrosomal vesicle and granule disappears in the fully formed acrosomal cap which shows a homogeneously PAS-positive intermediate layer. In the chimpanzee, the acrosomal vesicle makes a relatively very small contribution to the carbohydrate layer of the acrosomal cap which is mainly formed by spreading of material from the acrosomal granule.     

Leucocyte locomotion and chemotaxis : The influence of divalent cations and cation ionophores

Human blood neutrophil leucocytes and monocytes incubated in the absence of Ca²⁺ and Mg²⁺ showed reduced, but still substantial migration into micropore filters towards chemotactic agents, compared with cells migrating in a divalent cation-rich medium. This reduction in migration could be reversed by adding low doses of divalent cation ionophores (X537A or A23187) to the Ca²⁺- and Mg²⁺-free medium which suggests that migrating leucocytes in media depleted of extracellular divalent cations can make use of intracellular divalent cations and that the intracellular cation exchange necessary for locomotion is facilitated by the ionophores. At higher doses, the ionophores inhibited locomotion, as did procaine which reduces membrane permeability to cations. Little effect of K⁺ depletion or of ouabain on leucocyte locomotion was noted.     

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis

Summary The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes.The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O₂ ^– and H₂O₂, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O₂ ^– and H₂O₂ by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H₂O₂ is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H₂O₂ degrading enzymes in guinea pig cells.The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported.It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H₂O₂ degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.     

Antibody-mediated internalization of B lymphocyte surface membrane immunoglobulin

The ultrastructural correlates of antibody-mediated internalization of membrane immunoglobulin determinants has been studied in the guinea pig using a horseradish peroxidase conjugate of rabbit anti-guinea pig immunoglobulin. We have observed that polar flow of ligand-induced micro-aggregated membrane immunoglobulin proceeds by local deformation of the plasma membrane into open-ended endocytic vesicles which in turn accumulate at the Golgi-associated pole prior to completion of endocytosis. ‘Capping’ does not appear to result from simple diffusion of the aggregates within the plane of the membrane. In addition, the question of whether anti-immunoglobulin can induce the formation of uropods on B lymphocytes in the guinea pig was examined. Formation of uropod-like structures on B lymphocytes appears to be factitious and induced by accumulation at the Golgi-associated cell pole of non-internalizable aggregates of membrane immunoglobulin linked to cell debris. These observations are interpreted as supporting the concept that spontaneous uropod formation by lymphocytes in the absence of anti-immunoglobulin reagents is a characteristic of antigen-reactive thymus-derived lymphocytes in the guinea pig.     

The effects of concanavalin A on the mobility of lymphocyte surface receptors

It has been found that concanavalin A (Con A) bound to the lymphocyte surface can either induce cap formation or inhibit cap formation of various receptors including those for Con A itself. The expression of these antagonistic activities is highly dependent on the conditions under which cells are incubated with Con A. Incubation with Con A at 37 °C resulted in cap formation in only a small percentage of the cells and inhibited patch and cap formation induced by other reagents such as anti-immunoglobulin. In contrast, incubation of cells with Con A at 4 °C, followed by removal of unbound Con A molecules and elevation of the temperature to 37 °C resulted in cap formation in more than 40 % of the cells. Quantitative analyses suggest that these effects involve cross-linkage of Con A receptors, which occur in two states, mobile and relatively immobile. A model is proposed to explain the various effects of Con A in terms of the association of these receptors with colchicine binding proteins.     

Ribonuclease–gold Labels Chondroitin Sulphate in Guinea Pig Basophil Granules

Basophilic leucocytes are metachromatic granule-containing secretory granulocytes that contain a mixture of granular proteoglycans devoid of heparin. In guinea pigs, isolated basophilic leucocyte granules primarily contain chondroitin sulphate. We have recently demonstrated that an enzyme-affinity– gold technique to image RNA, using the reagent RNase gold, also binds specifically to heparin in human mast cell granules. Such binding is based on the known property of heparin as a competitive inhibitor of RNase. Using similar methods, we show here that RNase–gold binds to the chondroitin sulphate in the secretory granules of guinea pig basophils, thus broadening the applicability of this post-embedding affinity–gold method to studies that require imaging of chondroitin sulphate in routinely prepared electron microscopical samples.     

Immunochemical study of the leukocyte myeloperoxidases from various sources]

The antigenic difference between myeloperoxidases of human, rabbit, guinea pig, horse, dog, sheep and mouse leucocytes and horse radish peroxidase was investigated. By counterimmunoelectrophoresis with antiserum specific for human and mouse myeloperoxidase and horse radish peroxidase, the enzyme catalysing peroxidase reaction in leucocytes from the above sources was shown to possess species specificity and different antigenic composition.     

Macrophage metabolism: activation of NADPH oxidation by phagocytosis

Rabbit and guinea pig peritoneal and alveolar macrophages and rabbit polymorphonuclear leucocytes (PMN) have been tested for their capacity to oxidize NADPH and NADH. In all these cells granule-bound NADPH oxidase is much more active than NADH oxidase, thus confirming our previous observations on human blood and guinea pig PMN. If the phagocytes are challenged with bacteria, the activity of NADPH oxidase is considerably stimulated. The enhancement of the oxidase activity is due to an increase of its V_max and, in the case of the PMN, also to a decrease of the K_m. We conclude that NADPH oxidase might play a relevant role in the metabolic stimulation of both PMN and macrophages by phagocytosis.