Rabbit Polymorphonuclear Leukocytes Research Articles

Rabbit polymorphonuclear leukocyte migration in vitro in response to dental plaque.

Human dental plaque extracts in Gey's medium and whole human saliva were tested in the Boyden Chamber system for chemotactic activity against rabbit peritoneal polymorphonuclear leukocytes. Positive controls were a semipurified bacterial chemotactic factor and a complement derived chemotactic factor, Gey's medium alone served as a negative control. The method appeared to be very sensitive as concentrations of less than one mg plaque per ml medium gave a clear chemotactic response. Whole human saliva was less chemotactic than dental plaque. Incubation of equal concentrations of plaque extract in both the upper and lower compartments of the Boyden Chamber did not result in a large increase in cell migration, indicating that most cell migration in reaction to dental plaque extract in this system depends on a concentration gradient. The chemotactic factor or factors were present in plaque at the time it was collected, they were readily dissolvable in water, and they were heat stable. The dose range in which dental plaque extract would evoke a chemotactic response was variable and usually very narrow. Replicate tests. yielded highly variable results indicating that this method is not suitable for quantitative comparisons. Extracts of the same dilution were prepared from dental plaque samples from a small population with varying degrees of gingivitis. Differences in chemotactic response to those samples could not be related to the intensity of gingivitis.

Association of the alkaline phosphatase of rabbit polymorphonuclear leukocytes with the membrane of the specific granules.

The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated. The results obtained suggest very strongly that alkaline phosphatase is a component of the granule membrane. The enzyme remains attached to the membrane upon disruption of the granules by the use of detergents or by hypotonic shock and subsequent extraction with sodium sulfate, and can be isolated together with fragments of the granule membrane by isopycnic equilibration. Treatment of the granules with high amounts of Triton-X-100, sodium deoxycholate, or hexadecyltrimethylammonium bromide releases the enzyme in soluble form. In polymorphonuclear leukocyte homogenates, lysis of the granules is needed in order to render alkaline phosphatase fully accessible to substrates. This suggests that the catalytic site of the enzyme is exposed at the inner face of the granule membrane.

Phagocytosis of Salmonella typhimurium 395 MR10 by rabbit polymorphonuclear leucocytes: formation of phagolysosomes demonstrated by zonal separation.

After allowing phagocytic uptake of bacteria, rabbit polymorphonuclear leucocytes were disintegrated and fractionated by zonal centrifugation. Two different subcellular particles were obtained containing high relative activities of lysosomal marker enzymes. One particle displayed the sedimenting characteristics of lysosomes, the other sedimented with radio‐actively labelled bacteria subjected to phagocytosis. The properties of the latter particles and the conditions concerning their formation were those expected of phagolysosomes. The phagolysosomes showed a peak at a density = 1.19, whereas both lysosomes and nonphagocytosed bacteria were recovered at a density = 1.25. The results are discussed in relation to the analysis of host‐parasite interaction at a subcellular level.

The effect of adjuvant arthritis and drugs on the ability of rat plasma to inhibit the triton X-100 induced lysis of rabbit polymorphonuclear leucocyte granules

Plasma from normal rats has the ability to inhibit the Triton X-100 lysis of rabbits polymorphonuclear leucocyte granules. During the development of adjuvant-induced arthritis this activity decreases but no change occurs when rats are given injections of E. coli in oil into the foot pad. A study has been made of the levels of stabilizing activity present in the plasma of both normal and arthritic rats treated with either anti-inflammatory drugs or drugs alleged to affect the stability of lysosomal membranes. Paramethasone, prednisolone and menadione prevented the fall in levels during the onset of the adjuvant-induced arthritis but only paramethasone caused an increase in the protective effect when given to normal rats. Chloroquine, cortisone (lysosomal stabilizers) testosterone, oestradiol (lysosomal labilizers) progesterone and fenclozic acid failed to correct the fall in stabilizing activity in arthritic rats but progesterone, oestradiol and chloroquine caused a decrease in stabilizing activity when given to normal rats.

Disintegration of single cells in suspension. Isolation of rabbit polymorphonuclear leucocyte granules.

Disintegration of rabbit polymorphonuclear leucocytes in suspension is achieved by extrusion under controlled pressure. The operating pressure is the key factor influencing the degree of disintegration. The number of cells disintegrated increases with higher pressure. At 35 kp/cm2, 80–90 per cent of the cells are disintegrated. The disintegrated material was subsequently analysed by following the subcellular distribution of leucocyte A and B granule marker enzymes. The analysis reveals that although 80–90 per cent of the cells are disintegrated, a high degree of subcellular integrity remains. Leucocyte A and B granules, abundantly present in the disintegrated material, were separated from soluble components by zonal centrifugation. Extrusion under controlled pressure as a cell disintegration method is discussed, particularly with reference to the study of polymorphonuclear leucocyte structure and function.

NADPH oxidizing activity in rabbit polymorphonuclear leukocytes: Localization in azurophilic granules

Rabbit polymorphonuclear leukocyte granules were submitted to zonal fractionation through a discontinuous sucrose gradient. Distribution of azurophilic and specific granules, enzymatically characterized by peroxidase and alkaline phosphatase respectively, was as reported by others. NADPH oxidizing activity was associated with azurophilic granules. 3-Amino-1H-1, 2,4-triazole stimulated NADPH oxidation by azurophilic granules and inhibited peroxidase. Relationships between peroxidase and NADPH oxidizing activity are discussed.

Selective release of lysosomal hydrolases from phagocytic cells by cytochalasin B.

1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.

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.

Depression of 3 H-glucose uptake into rabbit polymorphonuclear leukocytes by glucocorticoids in concentrations partly saturating the specific glucocorticoid uptake. Evidence for a glucocorticoid receptor.

AbstractRabbit polymorphonuclear leukocytes have a specific glucocorticoid uptake in vitro, half saturated by cortisol at a probably physiological concentration (about 3 × 10‐9 M). These cells in vitro accumulate 3H‐d‐glucose and 3H‐2‐deoxy‐d‐glucose, possibly by a common uptake step. Cortisol, dexamethasone, 9αF‐prednisolone, prednisolone, triamcinolone and triamcinolone acetonide, all in the low concentrations sufficient to partly saturate the specific glucocorticoid uptake, depress the uptake of 3H‐glucose by these cells. Corticosterone acted as a partial agonist. 11‐epicortisol, tetrahydrocortisone and progesterone, which have no glucocorticoid activity, had a very low activity on 3H‐d‐glucose uptake. For 11‐epicortisol and tetrahydrocortisone this corresponded to a low affinity to the specific glucocorticoid uptake process, while progesterone had a relatively high affinity. Progesterone in high concentrations was shown to block the depressing effect on 3H‐d‐glucose uptake by cortisol, probably by keeping it away from the specific glucocorticoid uptake process. Rabbit polymorphonuclear leukocytes thus seem to have a specific glucocorticoid receptor.

Cartilage collagen: Inability to serve as a substrate for collagenases active against skin and bone collagen

Human gingival and rabbit polymorphonuclear leukocyte collagenases previously shown to be active against native monomeric skin and bone collagen, with the chain composition [α1(I)] 2α2, do not cleave native monomeric cartilage collagen, with the chain composition [α 1(II)] 3, as measured by a decrease in relative viscosity. Both types of collagen, however, are actively degraded by collagenase (EC 3.4.4.19) obtained from Clostridium histolyticum. The results strongly support the suggestion that collagen in cartilaginous structures is resorbed by a mechanism independent of that utilized in other tissues.

Induction of experimental emphysema. Cellular and species specificity.

Polymorphonuclear leukocytes from dogs, humans, and rabbits, and dog alveolar macrophages and dog mononuclear leukocytes were investigated for their ability to induce experimental emphysema in the dog. Eighteen dogs were treated with aerosolized homogenates of the various cell types or control substances. In decreasing order, the emphysema-inducing ability of the different cell homogenates was dog polymorphonuclear leukocytes, human polymorphonuclear leukocytes, dog alveolar macrophages, rabbit polymorphonuclear leukocytes, and dog mononuclear leukocytes. The last 2 cell types produced little or no disease. Proteinase assays were performed on homogenates of all cell types. The ability of the cell homogenates to induce experimental emphysema correlated with the presence of in vitro neutral and alkaline protease activity.

In-vitro activity of an antibacterial substance from rabbit polymorphonuclear leukocytes.

gen, the sugar residues (which confer 0 specificity and constitute the smooth, virulent side chains), and the stereochemical configuration between the cell-wall components all appear relatively unimportant for the expression of the cidal activity of the leukocytic extract. The possible presence of host antigens on fresh bacterial isolates and of serum in the in-vitro assay system, however, interfered with the cidal property. It is suggested that the cidal system of polymorphonuclear leukocytes may represent a defense mechanism in which a few related proteins could accomplish a number of tasks by interaction with a susceptible ligand on the unit bacterial cell wall. Bactericidal activity in extracts from polymorphonuclear leukocytes (PMNs) of several mam

Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis

Observations have been made on the inhibitory action of cytochalasin B (CB) on locomotion, phagocytosis and glycolysis in human, horse, and rabbit polymorphonuclear leucocytes (PMNs). Locomotion in all three species was suppressed by as little as 0.3 μg/ml CB and completely blocked by 3 μg/ml, as judged by direct observation or by a Millipore filter assay system. Phagocytosis was evaluated morphologically as well as in a bactericidal system. Three μg/ml CB suppressed, but did not completely block, phagocytosis by horse and human cells; increasing the concentration of CB to 40 μg/ml exerted little additional suppression of phagocytic capacity. Rabbit exudate cells ingested and killed certain species of bacteria Staph. aureus or Salmonella less effectively than did horse or human cells; in these instances CB did not alter the killing rate significantly CB did inhibit the killing of E. coli by rabbit cells. Three μg/ml CB resulted in a partial inhibition of several glycolytic parameters, i.e., glucose utilization, lactate production, and CO 2 production from C-1 or C-6 labeled glucose. These parameters were inhibited more than 75 % by 10 μg/ml CB. Studies made thus far indicate that CB probably suppresses glycolysis by interfering with glucose transport or phosphorylation. Comparison of the inhibition of various parameters by CB and by other metabolic inhibitors suggests that the glycolytic inhibition by CB is probably not the primary drug action responsible for the altered locomotion and phagocytosis.

Uptake of 3H-cortisol by rabbit polymorphonuclear leukocytes in vitro. III. Structural evidence for a specific glucocorticoid uptake

Rabbit polymorphonuclear leukocytes have a saturable 3H-cortisol uptake in vitro , half saturated around 3 × 10 −9M cortisol. The ability of 28 other non-radioactive steriods, mainly glucocorticoids, to depress the saturable 3H-cortisol uptake was also studied. Among glucocorticoids in clinical use triamcinolone acetonide and paramethasone were the most potent, 5–6 and 5 times cortisol respectively, while dexamethasone was only twice as potent as cortisol. The cortisol stereoismer, 11-epicortisol, which has no glucocorticoid activity in vivo , had very little depressing potency. For the group as a whole a highly significant rank correlation was found between the depressing potency and the in vivo glucocorticoid potency obtained from the literature (and determined in rats or mice). Rabbit polymorphonuclear leukocytes thus have a specific cortisol uptake, probably corresponding to an interaction with a glucocorticoid receptor.

Cytochemical localization of acid phosphatase activity in granule fractions from rabbit polymorphonuclear leukocytes.

When rabbit peritoneal exudates (97% polymorphonuclear [PMN] leukocytes, 2% mononuclear cells) were fractionated by zonal sedimentation or isopycnic centrifugation, four fractions (A, B, C, and D) were obtained, as reported earlier. "A" consisted largely of PMN azurophil granules, "B" of PMN specific granules, and "D" of membranous elements. The source of the more heterogeneous "C" fraction (containing acid hydrolases) was uncertain. To gain further information on the nature of this fraction, cytochemical tests for acid phosphatase (AcPase) were carried out on the starting cells and on the fractions. In intact PMN, lead phosphate reaction product was found in Golgi complexes, perinuclear cisternae, and some azurophil granules (immature forms or disrupted mature forms) of a few cells. The specifics and the intact azurophils were not reactive. Reaction product was also found within Golgi cisternae, secondary lysosomes, and some of the azurophil granules of mononuclear cells. Observations on the A and B fractions confirmed those in situ regarding the localization of reaction product in disrupted PMN azurophils, its absence from specifics, and the latency of the enzyme activity in intact azurophils. In the C fraction, AcPase was found in three structures (a) Golgi cisternae, (b) dense bodies, and (c) small pleomorphic granules Comparison with the starting cells indicates that the Golgi complexes are probably derived from both PMN leukocytes and mononuclear cells, whereas the remaining elements resemble (in size, shape, and density) secondary lysosomes and azurophil granules of mononuclear cells. The results indicate that the bulk of the cytochemically detectable AcPase present in the C fraction is derived from mononuclear cells, rather than from PMN leukocytes

The appearance of lecithin- 32 P, synthesized from lysolecithin- 32 P, in phagosomes of polymorphonuclear leukocytes.

Rabbit polymorphonuclear leukocytes ingesting paraffin oil particles stabilized with albumin, converted more lysolecithin-(32)P (added to the medium as an albumin complex) to cellular lecithin than did control cells. Almost all of the increment in leukocyte lecithin-(32)P is found in association with the isolated phagocytic vacuoles. About half of lecithin-(32)P of granulocytes incubated first with lysolecithin-(32)P and then reincubated with paraffin particles in a nonradioactive medium is transferred from a sedimentable (presumably membrane) fraction to the phagosomes. Isolated phagosomes or granules by themselves are capable of acylating lysolecithin. The main source of lysolecithin-(32)P for synthesis of cellular lecithin-(32)P, however, appears to be extracellular rather than lysolecithin-(32)P within the cytoplasm or the phagocytic vacuole. We interpret our findings therefore as indicating that lecithin-(32)P in the phagosomes derives chiefly from the outer membrane.

Studies on isolated membranes of azurophil and specific granules from rabbit polymorphonuclear leukocytes.

Membranes were prepared from rabbit polymorphonuclear leukocyte azurophil and specific granules separated by zonal differential centrifugation. The two types of granule membranes were quite similar in ultrastructural appearance, but they showed distinct differences in cholesterol-phospholipid ratios and in protein components demonstrable in polyacrylamide gels.

The inhibition of cytochrome oxidase by lysosomal cationic proteins of rabbit polymorphonuclear leukocytes

At 0.33 μM, a mixture of cationic proteins from PMN lysosomes decreased the velocity constant of rat liver cytochrome oxidase by 50%. Separation of the proteins into fractions of circa 8000 and 4000 M.W. reveals that the former is the more potent in its inhibition of cytochrome oxidase, causing 50% inhibition of activity at 0.1 μM.

Uptake of 3 H-cortisol by rabbit polymorphonuclear leukocytes in vitro. II. Effect of metabolic inhibitors and of temperature on uptake and washout.

AbstractRabbit pseudoeosinophilic polymorphonuclear leukocytes in vitro have an uptake of 3H‐cortisol, which is partly saturable and partly non‐saturable. The saturable component was much more dependent on temperature during accumulation and washout than the non‐saturable component. The metabolic inhibitors N‐ethyl maleimide, p‐chloro‐mercuribenzene sulfonic acid, iodoacetic acid and 2,4‐dinitro‐phenol and preheating at 67° C significantly decreased the saturable uptake while the non‐saturable uptake, if anything, was increased in some experiments. Dinitro‐phenol slightly accelerated the washout of the saturable component.The characteristics of the saturable 3H‐cortisol uptake strengthens the evidence that it corresponds to a specific interaction with a receptor in the rabbit pseudoeosinophilic polymorphonuclear leukocytes, since it behaves like the specific hormone uptake in other tissues under similar conditions. The non‐saturable uptake behaves like a non‐specific uptake.

Production of Chemotactic Activity by Guinea Pig Immunoglobulins Following Activation of the C3 Complement Shunt Pathway

Abstract Guinea pig γ1 and γ2 immunoglobulins consume late C components (C3 to C9) when incubated with C4 deficient guinea pig serum. The γ2 antibodies produce greater hemolytic activity losses in normal than in C4 deficient guinea pig serum. In contrast, consumption of late component activity by the γ1 antibodies is similar in serum containing or lacking C4. Both types of immunoglobulins produce a serumderived factor chemotactic for rabbit polymorphonuclear leukocytes in either normal or C4 deficient serum. This factor has been identified as a cleavage product of C5.