Differential role of neutrophils and monocytes during subcutaneous plasma extravasation

We examined the behavior of polymorphonuclear leukocytes (PMNs) and monocytes during subcutaneous plasma extravasation in guinea-pigs. Plasma extravasation was induced by intradermal injection of zymosan-activated plasma (ZAP). The degree of extravasation correlated logarithmically with the concentration of injected ZAP, and was composed of PMN-dependent and -independent components. The latter was mediated primarily by histamine. The former accounted for 40-50% of the total plasma extravasation, peaked within 15 min, and then rectilinearly decreased with a half-life between 30 and 40 min. Histological examination of skin at 15 min after ZAP injection demonstrated PMN attachment to the luminal surface of venule endothelial cells, without evidence of PMN extravasation. We next examined whether monocyte infiltration of subcutaneous tissue played a causal role in plasma extravasation. Monocyte-predominant infiltration was initially caused by an intradermal injection of a monocyte-specific chemotactic factor, the S19 ribosomal protein (RP S19) dimer. Monocyte infiltration did not induce plasma extravasation even in guinea-pigs with elevated peripheral blood monocyte levels following administration of a macrophage-colony stimulating factor. A simultaneous injection of prostaglandin E2, a vasodilating agent, with RP S19 dimer also did not induce plasma extravasation. In contrast, a simultaneous injection of RP S19 dimer with ZAP changed the leukocyte infiltration pattern from PMN-predominant to monocyte-predominant, and almost completely suppressed the PMN-dependent component of the ZAP-induced plasma extravasation. The lack of plasma extravasation in the monocyte-predominant pattern was reproduced when a strong monocyte infiltration was induced by an intradermal injection of apoptotic cells. We conclude that leukocyte-induced plasma extravasation is specific for PMN, and is not due to a physical leakage of plasma during leukocyte emigration. Rather, plasma extravasation is probably caused by a cognate interaction between PMNs and postcapillary venule endothelial cells.     

The modulation of enhanced vascular permeability by prostaglandins through alterations in blood flow (hyperemia)

The enhanced vascular permeability induced by histamine or bradykinin in the skin of the guinea-pig and rabbit was significantly augmented by small amounts of prostaglandins of the E type. When injected alone these prostaglandins had little effect on vascular permeability. Furthermore, E type prostaglandins were found to be more potent at inducing hyperemia than either histamine or bradykinin. Prostaglandin F₂ did not enhance the vascular permeability induced by histamine or bradykinin nor did it produce hyperemia in the skin. In the rat, prostaglandins alone enhanced vascular permeability but they also increased the effect of histamine, serotonin and bradykinin. Using⁸⁵Sr-microspheres to measure blood flow a correlation was found between the degree of hyperemia produced by prostaglandins and the degree to which they augmented enhanced vascular permeability due to histamine, serotonin or bradykinin. Prostaglandins therefore can directly mimic the hyperemia of the inflammatory process and can also modulate the changes in vascular permeability caused by other mediators of inflammation.Supported by grants MA-5046 and MT-1251 of the Medical Research Council of Canada and the Ontario Heart Foundation.     

Modification of neutrophil chemotactic responsiveness during various inflammatory reactions

Various inflammatory reactions have been induced in order to examine the chemotactic response of polymorphonuclear leucocytes (PMN) collected under various experimental conditions. Cells were harvested from the pleural cavity of rats after the induction of three acute non specific inflammatory reactions and two immune reactions. The results obtained with two techniques of chemotactic assessment (agarose assay and Boyden chamber technique) demonstrated a variation of chemotactic response depending on the cell source and the chemoattractants used. Using agarose assay, we distinguished locomotor reactivity of PMN harvested after immune inflammatory reactions from that of PMN harvested after non immune inflammatory reactions. Chemokinetic and chemotactic responses to various chemoattractants were inhibited in the first case and not affected in the second. Using the Boyden chamber technique, inhibition of random or oriented migration of PMN harvested after immune inflammatory reactions after the injection of a non antigenic irritant such as calcium pyrophosphate crystals (CaPP) was also observed.     

Prostaglandin activity in the course of immune specific and nonspecific inflammatory reactions in chickens

The presence of prostaglandins in inflammatory infiltration evoked by tuberculin and thermal factor in chickens was studied. The prostaglandin E2 activity was found in both specific and nonspecific immune inflammatory reactions in chickens. The authors discuss the role of prostaglandins as mediators in inflammatory processes.     

Suppression of nephrotoxic serum nephritis in rats by prostaglandin E1.

The ability of specific prostaglandins to modulate the development of nephrotoxic serum nephritis (NSN) in rats has been examined. The nephrotoxicity of one intravenous injection of antibodies directed against rat glomerular basement membrane (GBM) was markedly suppressed by treatment with a stable analog of prostaglandin E1 (15-(S)-15-methyl PGE1; 15-M-PGE1). Prostaglandin E1 treatment was shown to suppress both glomerular hypercellularity and proteinuria, while the binding of specific antibody to the GBM was not altered. These studies indicate that certain prostaglandins may play an important role in the regulation of Type II immune reactions. Further investigations of the role of arachidonic acid products undoubtedly will provide valuable information regarding the modulation of tissue injury as a result of various inflammatory reactions.     

Effect of Immune Serum or Polymyxin B on Escherichia coli-Induced Inflammation and Vascular Injury

Bacterial invasion of the tissues often stimulates a vigorous inflammatory reaction, which may limit the spread of microorganisms but may also be accompanied by serious vascular injury and tissue damage. We previously studied the inflammatory reaction induced by the injection of killed Escherichia coli into rabbit skin, a model suitable for the quantitation of various parameters of inflammation. Here we report the effect of immune serum treatment of the E. coli on their capacity to induce inflammation and vascular injury. Injection of killed E. coli treated with immune serum elicited a reaction which had a smaller increase in vascular permeability (protein exudation), measured with (125)I-labeled albumin, less increase in blood flow, measured with (86)RbCl, less leukocyte infiltration, measured with (51)Cr-labeled leukocytes, and a lesser degree of hemorrhage, measured with (59)Fe-labeled erythrocytes, than E. coli treated with nonimmune serum. Crossover experiments with four different E. coli serotypes and four different antisera indicated that antibody to specific O antigens or a related antigen, but not to K or H antigen, was important for modifying the inflammatory response. Treatment of four different E. coli serotypes with antiserum to "core" glycolipid, produced by immunization with the E. coli J5 mutant, inhibited the inflammatory response to all four E. coli serotypes. Finally, treatment of killed E. coli with polymyxin B also inhibited their inflammation-inducing potential. These results suggest that it may be possible to diminish the magnitude of local vascular and tissue injury associated with E. coli infections by the use of antisera or polymyxin B, which bind to endotoxin on the E. coli.     

Pulmonary and systemic vascular responses of perinatal goats to prostaglandins E1 and E2.

Effects of prostaglandins of the E series and their metabolites on pulmonary and systemic circulations of newborn and exteriorized fetal goats (anesthetized with chloralose) were evaluated in situ using an isolated perfused left lung lobe preparation. Prostaglandin E1 (PGE1) and, to a lesser extent, prostaglandin E2 (PGE2) infusions resulted in decreases of pulmonary vascular resistance (PVR) of fetal and neonatal goats. Infusions of PGE1 or PGE2 (less than 2 microgram.kg-1.min-1 for 1 min) directly into left pulmonary arterial blood did not affect systemic arterial pressure (SAP). Infusions of PGEs (greater than 2 microgram.kg-1.min-1 for 1 min) resulted in decreases in SAP and heart rate. The dose-response characteristics of the pulmonary circulation in response to PGE1 and PGE2 were not different in fetal and newborn goats. Fetal asphyxia did not alter the dose-response characteristics of pulmonary circulation in response to PGE1. Metabolites (15-keto) of PGE1 and PGE2 had no effect upon PVR or SAP of perinatal goats. These results demonstrate in perinatal mammals 1) vasodilator action of PGE1 and PGE2 on the pulmonary and systemic circulations, and 2) catabolism by the lungs of these prostaglandins.     

The in vivo effect of leukotriene B4 on polymorphonuclear leukocytes and the microcirculation. Comparison with activated complement (C5a des Arg) and enhancement by prostaglandin E2.

The effect of synthetic leukotriene B4 (LTB4) on chemotaxis in vivo (51Cr-polymorphonuclear leukocyte [PMN] accumulation) was examined and its potency compared with that of C5a des Arg-containing zymosan-activated plasma (ZAP). On a molar basis the amount of C5a des Arg calculated to be in our preparation of ZAP was found to be up to approximately 80 times more potent than LTB4, although in vitro the two chemotaxins have been reported to be about equipotent. ZAP is more representative of what may happen in vivo than its principal constituent C5a des Arg, but for a more precise comparison the purified and isolated peptide will have to be compared with synthetic LTB4. Whereas ZAP induced severe PMN-dependent microvascular injury (increase in vessel permeability [125I-albumin] and hemorrhage [59Fe-erythrocytes]), LTB4 only induced an increase in vascular permeability, and this occurred only in the presence of simultaneously injected prostaglandin E2 (PGE2). PGE2 also enhanced substantially the number of PMNs and the amount of exuded plasma at injection sites of the chemotaxins. However, unlike in two other reports, LTB4 did not cause an immediate transient increase in vessel permeability, nor did it enhance the permeability-increasing effect of bradykinin. Furthermore, unlike PGE2 LTB4 did not induce an increase in blood flow, but a decrease (57Co-microspheres). It is concluded that LTB4 may act as a host-derived chemoattractant in vivo, but, compared with that of ZAP (primarily activated complement), its role in acute inflammation is probably less significant than that of the complement-derived chemotaxin(s).     

Polymorphonuclear leukocyte accumulation in inflammatory dermal sites as measured by51Cr-labeled cells and myeloperoxidase

Two methods for quantitating polymorphonuclear leukocyte (PMN) accumulation in inflammatory skin lesions were studied. The lesions were produced in rats by intradermal injections of different dilutions of zymosan-activated plasma (ZAP). PMN accumulation in the skin lesions was estimated by determination of (1) homologous⁵¹Cr-labeled PMNs and (2) activity of myeloperoxidase (MPO) in the tissue sample.¹²⁵I-labeled human serum albumin was used for mesaurement of albumin extravasation. The [⁵¹Cr]PMN content and MPO activity in the skin lesions were both proportional to the concentration of ZAP injected. The correlation coefficient (r) between the two methods of measuring PMN accumulation in the inflammatory skin lesions was calculated to be 0.81±0.13 (mean±SD, N=8), The proportionality of the PMN accumulation to the different dilutions of injected ZAP, as measured both by [⁵¹Cr]PMN and by MPO activity, and the correlation of the two methods to each other, suggest that these two methods are reliable for measuring PMN accumulation in vivo. The inflammatory reaction also included albumin extravasation, which reached a relatively high level already at the lowest concentration of injected ZAP and did not seem to parallel PMN accumulation.     

Critically ill anergic patients demonstrate polymorphonuclear neutrophil activation in the intravascular compartment with decreased cell delivery to inflammatory focci.

Skin test anergy, the failure to produce a delayed type hypersensitivity (DTH) response, is associated with an increase in infection-related complications and death usually due to multiple organ failure (MOF). Refractory intravascular activation of polymorphonuclear neutrophils (PMNs) has been implicated in the development of MOF. We studied 20 critically ill surgical patients with life threatening infections to determine if PMN intravascular activation was present and how this affected essential PMN functions such as exudation. The 11 anergic patients had a more intense inflammatory response to their infection. Plasma lactoferrin was 6.1 +/- 0.3 microgram/ml in anergic patients compared to 3.9 +/- 1.5 in reactive P less than 0.05, accompanied by reduced total primary (3.3 +/- 1.9 vs 4.7 +/- 2.1 micrograms/10(6) PMN P less than 0.01) and secondary (2.8 +/- 0.4 vs 5.0 +/- 0.9 microgram/10(6) PMN P less than 0.01) granule content, respectively. In vitro superoxide production following 100 ng/ml PMA stimulation was 0.44 +/- 0.1 in anergics vs 0.36 +/- 0.1 nmol/microgram PMN protein in reactivities, P less than 0.05. PMN chemotaxis was 8.2 +/- 0.6 PMNs/HPF in anergics compared to 10.2 +/- 1.6 PMNs/HPF in reactives P less than 0.05, accompanied by decreased PMN delivery to skin blister windows (3.2 +/- 1.4 vs 4.5 +/- 1.9 x 10(7) PMN/ml, respectively, P less than 0.05). We conclude that critically ill anergic surgical patients have increased intravascular PMN activation, which may contribute to oxygen-derived tissue damage in the vascular space, as well as a deficient delivery of effector cells in areas of bacterial invasion. This may lead to inability to clear the inflammatory signals which set up the vicious circle of MOF leading to death.     

Acute inflammatory pulmonary reactions induced by chemotactic factors.

Acute inflammatory reactions have been produced in hamster lungs by the intrapulmonary instillation of preformed chemotactic mediators (C5fr and F-Met-Leu-Phe). By the use of 111Indium and 125Iodine labeling of homologous neutrophils (PMNs) and homologous albumin, respectively, it has been possible to obtain highly reproducible and quantitative parameters of the acute inflammatory response. The lung responds in a dose-dependent and time-dependent manner to the instillation of preformed chemotactic mediators. The quantitative parameters indicative of PMN influx were more prominent than changes in vascular permeability. The data obtained by the use of radiolabeled PMNs were confirmed by observation with light microscopy. Nonchemotactic substances such as human IgG, serum albumin, and C4 failed to induce inflammatory responses in lung. Interestingly, intact C5 instilled into lung was extremely phlogistic, apparently due to hydrolysis once within the lung. These studies provide an approach to reliable quantitative parameters of inflammatory reactions in the lung and emphasize the in vivo biologic effects of chemotactic mediators.     

The natural mediator for PMN emigration in inflammation: VI. Production of leucoegresin by inflammatory SH-dependent protease in active Arthus reactions in complement-depleted rabbits

A chemotactic factor (leucoegresin) specific for neutrophilic polymorphonuclear leucocytes was isolated from the site of active Arthus reactions in rabbits. This material was found to be associated with most of the chemotactic activity for the cells in the reactions. Its production by the neutral SH-dependent protease in the reactions was not related to the complement system. The effects of the protease and chemotactic factor were also unrelated to the complement system.

Most of the chemotactic activity for PMN leucocytes in active Arthus reactions in rabbits was associated with leucoegresin itself, because the chemotactic activity of inflammatory extracts was significantly absorbed by anti-leucoegresin. Production of this chemotactic substance by the inflammatory SH-dependent protease in the inflamed site was not related to the complement system, because a similar amount of leucoegresin was isolated from the same Arthus skin lesion or from the protease-induced skin lesion in rabbits whose serum complement was apparently depleted by an anti-complementary factor from cobra venom. The in vivo effects of the protease and leucoegresin were not influenced by such depletion of serum complement. It was thus indicated that leucoegresin played a significant and characteristic part in inflammatory leucotaxis. On the other hand, a slight decrease in the PMN emigration in active Arthus reactions in complement-depleted animals suggested the possible presence of a complement-derived chemotactic factor.

     

The effect of a single infusion of zymosan-activated plasma on the pulmonary microcirculation of sheep. Structure-function relationships

Activation of the complement cascade is one of the mechanisms through which endotoxin may cause acute lung damage. The structural and functional changes following infusion of complement-activated plasma are described. In five anesthetized open-chest sheep, the authors monitored pulmonary and systemic artery pressures for 1 hour before and for 4 hours following the start of zymosan-activated plasma (ZAP) infusion (2 ml/min over a 20-minute period). Cardiac output, blood gases, and the number of circulating white cells were also measured. In addition, we took lung biopsy tissue at baseline, 7.5, 15, 30, 60, 120, 180, and 240 minutes following the start of infusion. Lung lymph flow and protein concentration were also monitored in 2 sheep. Following ZAP infusion there was an early phase of leukopenia and marked pulmonary hypertension, followed by a phase characterized by a modest increase in the flow of protein-rich lung lymph. By light microscopy pulmonary sequestration of granulocytes was evident just 7.5 minutes following the start of ZAP infusion. Peripheral lung granulocytes increased threefold above control values by 7.5 minutes, increasing to sevenfold by 30 minutes. Electron-microscopic studies showed that some of the granulocytes were disrupted, and specific and azurophilic granules were seen in the lumen. By 15 minutes endothelial damage was apparent, and intravascular monocytes were surrounded by a proteinaceous coat. Edema accumulation and an infiltration of inflammatory cells in the lungs' connective tissue regions increased to 2 hours. From 2 hours, lung injury was less marked, and the number of peripheral lung granulocytes, fewer. Sequestration of granulocytes occurred with the onset of pulmonary hypertension and leukopenia, and was most marked when lung injury was most severe. Transient endothelial damage and edema preceded the physiologic changes interpreted as an increase in pulmonary vascular permeability. Although pulmonary sequestration of granulocytes was at least as great as that with endotoxemia, unlike endotoxemia, ZAP caused only transient endothelial injury and modest changes in vascular permeability.     

Early T cell response to allografts occurring prior to alloantigen priming up-regulates innate-mediated inflammation and graft necrosis

The early inflammatory response within organ allografts is initiated by ischemia/reperfusion (I/R) and promotes subsequent alloantigen-primed T cell recruitment into and rejection of the graft. Polymorphonuclear leukocyte (PMN)-mediated tissue damage is a primary component of the early inflammation in allograft rejection. We sought to compare and elucidate the mechanism of early PMN infiltration into cardiac isografts and allografts. Despite identical production of PMN attractant chemokines, PMN infiltration following reperfusion into syngeneic and allogeneic grafts was not equivalent. PMN infiltration into isografts peaked at 9 to 12 hours post-transplant and quickly resolved. In contrast, PMN infiltration into allografts continued to elevated levels, peaking at 24 hours post-reperfusion. This amplified PMN infiltration into allografts did not resolve until 72 hours post-reperfusion and was accompanied by marked parenchymal necrosis. This early innate inflammatory response was regulated by IFN-gamma-producing CD8+ T cells present in the recipient before detectable alloantigen T cell priming. Co-culture with CD62L(low) CD8+ T cells, but not CD62L(high) CD8+ or CD62L(low) CD4+ T cells, harvested from na?ve animals induced allogeneic endothelial cells to express IFN-gamma-dependent chemokines. These data demonstrate CD8+ T cell-mediated attack on the vascular endothelium of allografts within hours following organ reperfusion that amplifies innate immune-mediated intra-graft inflammation and necrosis.     

Effect of prostaglandins E<Subscript>2</Subscript> and D<Subscript>2</Subscript> on presynaptic NMDA receptors in rat cerebral cortex

We studied the effect of prostaglandins on presynaptic NMDA receptors. Prostaglandin E₂ inhibited NMDA-induced ⁴⁵Ca²⁺ uptake by synaptosomes in low concentrations (IC₅₀ ∼10 μM), but potentiated it in higher concentrations. Prostaglandin D₂ increased ⁴⁵Ca²⁺ uptake by synaptosomes during stimulation of NMDA receptors. Our results indicate that prostaglandins D₂ and E₂ modulate function of presynaptic NMDA receptors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 271–273, September, 2007     

Prostaglandins and inflammatory cell movement in vitro

The prostaglandins PGE1, E2, F2alpha, and A1, had no significant cell migration inhibitory or chemotactic activity for guinea pig peritoneal exudate cells when tested in vitro using respectively the capillary tube cell migration test and the Boyden chamber test for chemotaxis. PGE1 and PGE2 also did not affect the positive chemotactic action of lymphokine. The proposed role of prostaglandins as chemotactic agents in inflammatory responses therefore should be treated with some caution.     

3-deaza-adenosine inhibition of stimulus-response coupling in human polymorphonuclear leukocytes

In an effort to define better the functional role of S-adenosyl-methionine-mediated methylation reactions in modulating polymorphonuclear (PMN) functional responses to chemotactic stimuli, we investigated the effects of 3-deaza-adenosine (3-DZA), a known inhibitor of methylation reactions in phagocytic cells, on formyl methionyl-leucyl-phenylalanine (FMLP)-induced responses in human PMN leukocytes. Using the fluorescent cyanine dye 3,3'-dipropylthiocarbocyanine (di-S-C3-(5)) as an optical probe of membrane potential we observed that 3-DZA at concentrations that inhibit FMLP-induced O2- production does not significantly alter FMLP-induced changes in transmembrane potential. Additional studies showed an inhibitory effect of 3-DZA on FMLP-induced PMN pinocytosis and to a lesser degree on FMLP-induced degranulation. However, pretreatment of PMNs with 3-DZA did not alter FMLP-induced changes in Quin-2 fluorescence, an indicator of changes in intracellular calcium levels. These findings demonstrate a dissociation between chemotactic factor-induced cell membrane depolarization, changes in intracellular calcium, and specific neutrophil functional responses and suggest that chemotactic factor-induced changes in transmembrane potential and intracellular calcium are independent of chemotactic factor-induced methylation reactions. Furthermore, 3-DZA did not alter phorbol myristate acetate induced O2- production or fluid pinocytosis indicating a stimulus specificity for the inhibitory effects of this agent on O2- production.     

Functional properties of a novel neutrophil chemotactic factor derived from human monocytes

Neutrophilic granulocytes (PMN) are attracted to sites of inflammation by chemotactic factors, the most potent of which are the complement split product C5a, the leukotriene B4 and the bacterial chemotactic factor-related tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP). In addition to inducing directed migration, these agents increase the adherence of PMN to synthetic surfaces and endothelial cells; some stimulate an oxidative burst and the production of reactive oxygen derivatives, and they may be involved in the release of granule constituents. Here, we describe studies on the activities stimulated by a novel monocyte-derived chemotaxin (MOC). Human MOC attracted human PMN, but not monocytes or eosinophils. Like all chemotactic agents, it increased the adherence of PMN on nylon fibers. In contrast to other chemotactic factors it did not stimulate the release of superoxide anion regardless whether the cells were in suspension or adherent on nylon fibers. There was no release of the primary granule enzyme glucosaminidase or the secondary granule component vitamin B12-binding protein in the absence or presence of cytochalasin B. The results suggest that MOC is a unique chemotactic agent with properties different from the most potent chemotactic factors C5a, LTB4 and FMLP. The delayed release from macrophages suggests its involvement in protracted and chronic inflammatory reactions.     

Relationship between neutrophil-mediated oxidative injury during acute experimental pyelonephritis and chronic renal scarring.

Previous experiments with rats have suggested that pyelonephritic scarring after acute ascending Escherichia coli pyelonephritis partly results from excessive polymorphonuclear leukocyte (PMN) infiltration and activation in the kidney parenchyma. We have studied the role of PMN oxidative metabolism in generating tissue injury during acute pyelonephritis. Rats with acute pyelonephritis were treated with dapsone (25 mg/kg twice daily for 3 days), a compound known to prevent PMN oxidant damage. In vitro, levels of dapsone easily achieved in vivo inhibited myeloperoxidase (MPO)-mediated reactions involving the oxidation of halides to reactive cytotoxic hypohalites (such as MPO-mediated iodination and luminol-enhanced chemiluminescence). In contrast, dapsone had no effect on superoxide production, lysosomal enzyme release, or bacterial killing by activated PMN. In vivo, dapsone treatment had no significant effect on acute pyelonephritis with respect to (i) bacterial counts, (ii) inflammatory swelling, and (iii) PMN infiltration. However, dapsone-treated animals sacrificed 2 months after acute pyelonephritis had a 65% reduction of renal scars when compared with controls. Since dapsone had no antibacterial effect, this protection is compatible with the hypothesis that dapsone prevented oxidant-generated tissue injury due to the extracellular release of the MPO system by activated PMN during acute suppurative pyelonephritis.     

The natural mediator for PMN emigration in inflammation: III. In vitro production of a chemotactic factor by inflammatory SH-dependent protease from serum immunoglobulin G

In earlier work, a chemotactic factor (leucoegresin) specific for polymorphonuclear (PMN) leucocytes had been isolated from the sites of Arthus reactions or cutaneous burns. The substance shared antigenic sites with IgG.

The possible existence precursor of the chemotactic factor in the γ₂-globulin fraction of normal rabbit sera is suggested since the protein fraction on incubation with a purified neutral SH-dependent protease from inflammatory tissue became strongly chemotactic.