CvL, a lectin from the marine sponge Cliona varians: Isolation, characterization and its effects on pathogenic bacteria and Leishmania promastigotes

CvL, a lectin from the marine sponge Cliona varians was purified by acetone fractionation followed by Sepharose CL 4B affinity chromatography. CvL agglutinated papainized treated human erythrocytes with preference for type A erythrocytes. The lectin was strongly inhibited by monosaccharide d-galactose and disaccharide sucrose. CvL is a tetrameric glycoprotein of 28 kDa subunits linked by disulphide bridges with a molecular mass of 106 kDa by SDS-PAGE and 114 kDa by Sephacryl S300 gel filtration. The lectin was Ca2+ dependent, stable up to 60 degrees C for 60 min, with optimum pH of 7.5. CvL displays a cytotoxic effect on gram positive bacteria, such as Bacillus subtilis and Staphylococcus aureus. However, CvL did not affect gram negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Leishmania chagasi promastigotes were agglutinated by CvL up to 2(8) titer. These findings are indicative of the physiological defense roles of CvL and its possible use in the antibiosis of bacteria and protozoa pathogenic.     

Pro-inflammatory effect of Arum maculatum lectin and role of resident cells

Arum maculatum agglutinin (AMA) is a monocot lectin isolated from tubers of Arum maculatum L. (Araceae) which exhibits different specificity towards oligo-mannosidic-type and N-acetyllactosaminic-type glycans. We have investigated the effect of this lectin on the cells of the immune system. Models of neutrophil migration in vivo, neutrophil chemotaxis in vitro and macrophage cultures were used to study the lectin inflammatory activity. When administered into rat peritoneal cavities, AMA (80, 200 and 500 microg/mL/cavity) induced significant and dose-dependent neutrophil migration. This effect was inhibited by incubation with alpha-methyl-d-mannoside. A 83% depletion in the number of resident cells following peritoneal lavage did not reduce the AMA-induced neutrophil migration, as compared to sham animals (not washed). However, pre-treatment with 3% thioglycolate which increases the peritoneal macrophage population by 236%, enhanced the neutrophil migration induced by AMA (200 microg/mL/cavity) (119%, p < 0.05). Reduction of peritoneal mast cell population by chronic treatment of cavities with compound 48/80 did not modify AMA-induced neutrophil migration. The neutrophil chemotaxy assay in vitro shows that the lectin (300 microg/mL) induces neutrophil chemotaxy (368% p < 0.05) compared to RPMI. Finally, injection into peritoneal cavities of supernatants from macrophage cultures obtained after stimulation with AMA (300 microg/mL) enhanced neutrophil migration (110% p < 0.05). Summarizing, our data suggest that A. maculatum agglutinin presents pro-inflammatory activity, inducing neutrophil migration by two ways, one which is independent on resident cells and another one dependent on the presence of these cells.     

Neutrophil recruitment inhibitory factor: a possible candidate for a novel cytokine

Inhibitory effect upon neutrophil migration to the inflammatory focus was previously detected in the cell-free incubation fluid of lipopolysaccharide (LPS)-stimulated macrophage monolayers. In the present study we showed that the neutrophil recruitment inhibitory activity from this supernatant was mainly detected in a fraction (P2) obtained by gel filtration chromatography on Sephacryl S-300. P2 fraction was able to inhibit 'in vivo' neutrophil emigration induced by different inflammatory stimuli, but it did not affect 'in vitro' neutrophil chemotaxis induced by FMLP. When injected intravenously, P2 inhibited oedema induced by carrageenin or immunological stimulus but not the oedema induced by dextran, thus affecting cell-dependent inflammatory responses. It was observed that P2 also induced neutrophil migration when injected locally in peritoneal cavities. This activity was significantly reduced by pretreatment of the animals with dexamethasone. Cytokines, such as IL-8 and TNF-alpha that are known to exhibit inhibitory effect upon neutrophil migration, were not detected in P2 fraction by highly sensitive assays. Overall the results suggest the existence of a novel cytokine exhibiting 'in vivo' neutrophil inhibitory activity, referred as NRIF.     

Anti-inflammatory effect of glucose-mannose binding lectins isolated from Brazilian beans

Selectins are essential for leukocyte recruitment in inflammation. Because of a lectin domain present in the selectin structure, we investigated the anti-inflammtory activity of six mannose-glucose binding lectins from brazilian beans: Dioclea guianensis-DguiL; D. grandiflora-DgL; Cratylia floribunda-CfL; D. violacea-D.vL; D. virgata-DvirL and Canavalia brasiliensis-ConBr. The lectins were injected intravenously (i.v.) into rats (0.1 and 1.0 mg/kg; 30 min before irritants) and its activities compared to E. coli endotoxin (LPS,30 mug/kg i.v.). Three lectins (DvL, CfL and DguiL), although less intense than LPS, inhibited the neutrophil migration induced by carrageenan (Cg, 300 mug) in a dose-dependent manner (0.1 and 1.0 mg/kg). DvL activity was reversed by 0.1 M alpha-D-methyl-mannoside (alpha-CH3), but not by 0.1 M alpha-D-galactose. The fMLP (44 ng)-induced neutrophil migration was also reduced by these lectins. Endotoxin contamination of lectin samples could be excluded since alpha-CH3 treatment reversed the DvL effect, but did not modify LPS inhibitory activity. Carrageenan (300 mug)-induced paw oedema was also reduced by LPS or lectin treatments. Conversely, none of the tested lectins inhibited dextran (Dex, 300 mug)-induced paw oedema, a classical leukocyte independent model, or zymosan (Zy, 1.0 mg)-induced peritonitis and paw oedema. LPS showed no effect upon Dex-induced paw oedema and barely reduced (25%) the oedematogenic effects of zymosan. As proposed for LPS, the lectin inhibitory activity was better observed on neutrophil-mediated inflammatory reactions. We speculate that the plant lectin antiinflammatory activity is probably due to a competitive blockage of a common leukocyte and/or endothelial selectin carbohydrate ligand.     

Effect of astasilid on the stimulation of peritoneal macrophages

The effect of astasilid, a sucrose monoester and the effect of mainly unsaturated fatty acids from the lipid fraction of Astasia longa on immunocompetent cells--macrophages of the mouse peritoneal cavity were studied. It was shown that astasilid increased 7.5-8.5-fold expression of Fc-receptors on the macrophage plasmic membranes and stimulated 5.5-6.5-fold the macrophage capacity for Fc-dependent phagocytosis of sheep red blood cells. Astasilid had no effect on migration of the macrophages into the abdominal cavity.     

Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J2, reduces neutrophil migration via a nitric oxide pathway

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ2-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ2 administration decreases leukocyte rolling and adhesion to the inflamed mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS-/- mice were not susceptible to 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ2-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-1 expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ2, whereas 15d-PGJ2 inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ2 suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.     

Conceivable difference in the anti-inflammatory mechanisms of lipocortins 1 and 5

Human recombinant lipocortins (LCT) 1 and 5 have been expressed in a yeast secretion vector and purified by ion exchange chromatography. The action of the proteins has been investigated in two models of experimental acute inflammation in the rat: carrageenin induced paw oedema and zymosan induced pleurisy. The effects of the proteins on PGE(2) release in vitro by rat macrophages stimulated with zymosan and on rat neutrophil chemotaxis induced by FMLP have also been assessed. LCT-1 significantly inhibited both paw swelling in carrageenin oedema and leukocyte migration in zymosan pleurisy. Moreover it showed a dose dependent, inhibitory effect on PGE(2) release. Neutrophil chemotaxis was only weakly affected by LCT-1. Conversely LCT-5 did not reduce carrageenin oedema and slightly inhibited PGE(2) release, but showed profound, dose dependent inhibitory activity on leukocyte migration in zymosan pleurisy and on neutrophil chemotaxis. These data suggest that LCT-1 acts mainly by interfering with arachidonic acid metabolism via the inhibition of phospholipase A(2). The anti-inflammatory activity of LCT-5, at variance with LCT-1, may be due to a direct effect on cell motility in addition to the interference with arachidonic acid metabolism.     

The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo

The third member of the family of junctional adhesion molecules (JAMs), JAM-3, also called JAM-C, was recently shown to be a novel counter-receptor on platelets for the leukocyte beta(2)-integrin Mac-1 (alphaMbeta(2), CD11b/CD18). Here, new functional aspects of the role of endothelial cell JAM-C were investigated. Endothelial cells express JAM-C, which is predominantly localized within junctions at interendothelial contacts, since it codistributes with a tight junction component, zonula occludens-1. Whereas JAM-C does not participate in neutrophil adhesion to endothelial cells, it mediates neutrophil transmigration in a Mac-1-dependent manner. In particular, inhibition of JAM-C significantly reduced neutrophil transendothelial migration, and the combination of JAM-C and platelet/endothelial cell adhesion molecule-1 blockade almost completely abolished neutrophil transendothelial migration in vitro. In vivo, inhibition of JAM-C with soluble mouse JAM-C resulted in a 50% reduction of neutrophil emigration in the mouse model of acute thioglycollate-induced peritonitis. Thus, JAM-C participates in neutrophil transmigration and thereby provides a novel molecular target for antagonizing interactions between vascular cells that promote inflammatory vascular pathologies.     

The neutrophil migration induced by tumour necrosis factor alpha in mice is unaffected by glucocorticoids

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFalpha), IL-8, C5a and leukotriene B(4) (LTB(4)). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulated in vitro with LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFalpha. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFalpha was also unaffected by pretreatment of the mice with dexamethasone. Moreover, neutrophil migration induced by intraperitoneal injection of LPS was completely blocked by pretreatment of the mice with a monoclonal antibody against murine TNFalpha. In conclusion, our results support the hypothesis that, in contrast to the role of TNF in rats (where it indirectly induces neutrophil migration), in mice, it may be an important mediator in the recruitment of neutrophils to inflammatory sites.     

Helianthus tuberosus agglutinin directly induces neutrophil migration, which can be modulated/inhibited by resident mast cells.

We investigated the effect of Helianthus tuberosus agglutinin (HTA) on neutrophil migration in vivo and in vitro. The role of resident cells in this effect was analyzed. Peritonitis was induced by injecting stimuli into rat (150-200 g) peritoneal cavities, and in vitro neutrophil chemotaxis was performed using a Boyden microchamber. HTA (80, 200, or 500 microg/mL per cavity) induced significant in vivo neutrophil migration (p < 0.05); in vitro assays showed that this lectin also induced neutrophil chemotaxis, an effect inhibited by the incubation of lectin associated with alpha-D(+)-mannose, its specific binding sugar. Depletion of the resident-cell population by peritoneal lavage did not alter HTA-induced neutrophil migration (200 microg/mL per cavity). The opposite strategy, increasing peritoneal macrophages by intraperitoneally injecting rats with thioglycollate, did not enhance the neutrophil migration produced by HTA (200 microg/mL per cavity). In addition, injection of supernatant from HTA-stimulated macrophage culture (300 microg/mL) into rat peritoneal cavities did not induce neutrophil migration. However, reduction of the peritoneal mast-cell population potentiated the neutrophil migration (p < 0.05) induced by HTA (200 microg/mL per cavity). Lectin from H. tuberosus has a direct neutrophil chemotatic effect that is modulated by mast cells.     

N-Amino acid linoleoyl conjugates: anti-inflammatory activities

Several N-linked amino acid-linoleic acid conjugates were studied for their potential as anti inflammatory agents. The parent molecule, N-linoleoylglycine was tested in an in vivo model, the mouse peritonitis assay where it showed activity in reducing leukocyte migration at doses as low as 0.3mg/kg when administered by mouth in safflower oil. Harvested peritoneal cells produced elevated levels of the inflammation-resolving eicosanoid 15-deoxy-Δ(13,14)-PGJ(2). These results are similar to those obtained in earlier studies with N-arachidonoylglycine. An in vitro model using mouse macrophage RAW cells was used to evaluate a small group of structural analogs for their ability to stimulate 15-deoxy-Δ(13,14)-PGJ(2) production. The d-alanine derivative was the most active while the d-phenylalanine showed almost no response. A high degree of stereo specificity was observed comparing the d and l alanine isomers; the latter being the less active. It was concluded that linoleic acid conjugates could provide suitable templates in a drug discovery program leading to novel agents for promoting the resolution of chronic inflammation.     

Macrophage migration inhibition by T-cells in rats after overcoming natural tolerance to alpha fetoprotein]

Cellular immunity to alpha-fetoprein (AFP) was studied in rats by the macrophage migration inhibition (MMI) test after breakage of tolerance to homologous AFP of rat (RAFP). A single injection of cross-reacting AFP of mouse (MAFP) has induced the capacity of peritoneal exudate cells of rats to react in the MMI test to both MAFP and RAFP. The second injection of MAFP results in reduction of the MMI reaction to both antigens and the appearance of stimulation in the macrophage migration together with further elevation of antibody titer to MAFP and RAFP. The fractionation of rat peritoneal cells has shown the T-cell nature of MMI reaction to both MAFP and RAFP.     

Stimulation of TNF-alpha, IL-1beta and nitrite release from mouse cultured spleen cells and lavaged peritoneal cells by mastoparan M

Chemically synthesized mastoparan M, a tetradecapeptide toxin of venom (INLKAIAALAKKLL), was used in the experiments described. After addition of mastoparan M to cultures of mouse macrophages in vitro, tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) were detected in the culture fluids by 12 h and their highest accumulation was observed by 24 h. Mastoparan M induced increases in both TNF-alpha secretion and mRNA level at the same time. Nitrite levels, which reflect nitric oxide synthesis, were also found to increase in the macrophage cultures at 24 h after mastoparan M addition. In vivo studies showed that mastoparan M induced the formation and accumulation of TNF-alpha, IL-1beta and nitrite in the peritoneal exudates of mice much faster at 90 min, 120 min and 180 min after mastoparan M injection, respectively. Similarly, significant increases in myeloperoxidase activity, a marker for neutrophil and macrophage content, were observed in the peritoneal lavage cells after intraperitoneal injection of mastoparan M. However, induction of nitrite by mastoparan M was completely inhibited by simultaneous addition of antimouse TNF-alpha antibody to the macrophage cultures. These results suggest that modulation of both neutrophil and macrophage influx by mastoparan M may be conveyed through TNF-alpha and IL-1beta secretion accompanied by nitrite formation.     

Stimulation of phagocytic function by factors inhibiting leukocyte and macrophage migration in humans

Fractions containing macrophage migration inhibition factor (MIF) and leucocyte migration inhibition factor (LIF) were obtained using Sephadex G-200 filtration from supernatant fluids of human lymphocyte cultures stimulated by PHA. The fractions were tested for the ability to affect migration and phagocytic activity of target cells. Peripheral blood leucocyte migration capacity was inhibited by the fraction with the molecular mass of 60,000-70,000 D (LIF), while migration activity of mouse peritoneal exudate cells was suppressed by the fraction with the molecular mass of 20,000-30,000 D (MIF). MIF- and LIF-containing fractions increased almost three-fold Fc-receptor-mediated phagocytic activity of neutrophils.     

Dexamethasone inhibits leukocyte migration through endothelial cells towards smooth muscle cells

Leukocyte interactions with endothelial cell monolayers (ECM) and smooth muscle cells (SMC) play an important role during inflammatory processes. Several studies describe an inhibitory effect of dexamethasone on polymorphonuclear leukocytes (PMNL), endothelial cell function, and interleukin-1 (IL-1) release. Aim of the current study was to investigate the influence of dexamethasone on leukocyte migration through an endothelial cell monolayer towards SMC-layers stimulated by tumor necrosis factor-alpha (TNF-alpha). Using a recently developed triple chamber migration system, SMC-layers were cultured on the bottom of a 24-well plate. On the upper surface of the first filter, ECM were cultured, the second filter was a collecting filter. The amount of leukocyte migration through ECM towards TNF-alpha-stimulated smooth muscle cell layers with and without dexamethasone-pretreatment was measured using a fluorescence technique. The pretreatment of SMC-layers with dexamethasone reduced the amount of leukocyte migration down to 92 +/- 8.8% (0.001 mM, p=n.s.), to 67 +/- 5.7% (0.01 mM, p<0.05), to 53 +/- 4.6% (0.1 mM, p<0.05), and to 41 +/- 5.0% (1 mM, p<0.05). In conclusion, dexamethasone treatment of smooth muscle cell layers inhibits leukocyte migration through ECM towards smooth muscle cell layers. The inhibition seems to be due to a decrease in IL-1 release. Treatment of all cell types, PMNL, endothelial cells, as well as smooth muscle cell layers, simulating an in-vivo situation, seems to have an additive effect.     

Cutting edge: Fc receptor type I for IgG on macrophages and complement mediate the inflammatory response in immune complex peritonitis

The contributions of Fc receptors (FcRs) for IgG (FcgammaRs) and complement to immune complex (IC)-mediated peritonitis were evaluated in BALB/c-, C57BL/6-, FcRgamma chain-, and FcR type III for IgG (FcgammaRIII)-deficient mice, backcrossed to the C57BL/6 background. In BALB/c mice, but not in C57BL/6 mice, neutrophil migration was markedly attenuated after complement depletion. In mice lacking FcRgamma chain, neutrophil migration was abolished, whereas it was unaffected in FcgammaRIII-deficient mice. Huge amounts of TNF-alpha (TNF) were found in the peritoneal exudate of BALB/c and C57BL/6 mice but were absent in mice lacking FcRgamma chain or FcgammaRIII. Surprisingly, a functional inhibition of TNF in BALB/c and C57BL/6 mice had no effect on neutrophil infiltration. These data provide evidence that in IC peritonitis, the activation of FcR type I for IgG on peritoneal macrophages and the activation of the complement cascade, but not the interaction of ICs with FcgammaRIII and the subsequent release of TNF, initiate the inflammatory response in BALB/c and C57BL/6 mice.     

Effects of some isoxazolpyrimidine derivatives on nitric oxide and eicosanoid biosynthesis

The inhibitory effect of some isoxazolpyrimidine derivatives on iNOS and COX-2 endotoxin induction in mouse peritoneal macrophages has been studied. Three of these compounds inhibited nitrite and PGE2 accumulation in a concentration dependent-manner at microM range. None of these active compounds affected iNOS, COX-2, COX-1 or PLA2 activities, although some reduced iNOS or COX-2 expression. Besides, no effect was observed on human neutrophil inflammatory responses (LTB4 biosynthesis and superoxide or elastase release). Active compounds were assayed by oral administration in the mouse air pouch model, where they inhibited nitrite accumulation without affecting PGE2 levels or leukocyte migration.     

Leukotriene B4 mediates neutrophil migration induced by heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.     

Decreased exhaled nitric oxide as a marker of postinsult immune paralysis.

Nitric oxide (NO) regulates neutrophil migration and alveolar macrophage functions such as cytokine synthesis and bacterial killing, both of which are impaired in immune paralysis associated with critical illness. The aim of this study was to determine whether NO is involved in immune paralysis and whether exhaled NO measurement could help to monitor pulmonary defenses. NO production (protein expression, enzyme activity, end products, and exhaled NO measurements) was assessed in rats after cecal ligation and puncture to induce a mild peritonitis (leading to approximately 20% mortality rate). An early and sustained decrease in exhaled NO was found after peritonitis (from 1 to 72 h) compared with healthy rats [median (25th-75th percentile), 1.5 parts per billion (ppb) (1.2-1.7) vs. 4.0 ppb (3.6-4.3), P < 0.05], despite increased NO synthase-2 and unchanged NO synthase-3 protein expression in lung tissue. NO synthase-2 activity was decreased in lung tissue. Nitrites and nitrates in supernatants of isolated alveolar macrophages decreased after peritonitis compared with healthy rats, and an inhibitory experiment suggested arginase overactivity in alveolar macrophages bypassing the NO substrate. Administration of the NO synthase-2 inhibitor aminoguanidine to healthy animals reproduced the decreased neutrophil migration toward alveolar spaces that was observed after peritonitis, but L-arginine administration after peritonitis failed to correct the defect of neutrophil emigration despite increasing exhaled NO compared with D-arginine administration [4.8 (3.9-5.7) vs. 1.6 (1.3-1.7) ppb, respectively, P < 0.05]. In conclusion, the decrease in exhaled NO observed after mild peritonitis could serve as a marker for lung immunodepression.     

Mechanism of action of an exopolysaccharide from Mycobacterium cyaneum on the local cellular reaction in experimental coli infection

A study was made of the effect of the exopolysaccharide (PS) M. cyaneum B-646 on cellular reaction in peritoneal exudate of white mice infected with E. coli. PS intensified the migration of phagocytic cells to the focus of infection, accelerated neutrophil maturation, promoted an earlier and more active involvement of neutrophils, particularly of macrophages, into the phagocytic process. In this case, there was a marked correlation between the magnitude of phagocyte population (of macrophage population to a greater degree) in peritoneal exudate and absorption capacity. PS activated macrophagal lysosomes, affecting selectively the accumulation of enzymes by the cells and lysosomal membrane permeability. The action of PS is dose-dependent. Under experimental conditions in question, the most favourable effect on local cellular reaction was exerted by PS in a dose of 20 micrograms per mouse.