Isolation and partial chemical characterization of macrophage-derived neutrophil chemotactic factor

Macrophages stimulated with lipopolysaccharide (LPS) release a factor (MNCF; macrophage-derived neutrophil chemotactic factor) which induces neutrophil migration in vivo and in vitro. The in vivo chemotactic activity of crude MNCF is not affected by pretreating the animals with dexamethasone, an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. We purified MNCF by affinity chromatography of the supernatant from LPS-stimulated macrophages on immobilized D-galactose, followed by gel filtration of the sugar-binding material on Superdex 75. The activity was eluted in the volume corresponding to a MW of 54 kDa. SDS-PAGE of this preparation revealed a single band, also corresponding to a 54 kDa protein. MNCF is an acidic protein (pI < 4) as shown by chromatofocussing. Like the crude MNCF, the homogeneous protein induced neutrophil migration in vitro as well as in vivo. This was not modified by dexamethasone pretreatment.     

Biological characterization of purified macrophage-derived neutrophil chemotactic factor

We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF). This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS). In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose-response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP) or interleukin 8 (IL-8), the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis) in the inflamed tissues.     

Tumor necrosis factor alpha (TNFalpha)-like factor produced by macrophages in rainbow trout, Oncorhynchus mykiss

The presence of a Tumor Necrosis Factor alpha (TNFalpha)-like molecule has been suggested in fish by biological assays and biological and antigenic cross-reactivities with human TNFalpha. In the present study, whether rainbow trout macrophages produce TNFalpha was examined. Murine recombinant TNFalpha (m-rTNFalpha) was used as the standard mammalian TNFalpha. The supernatants were harvested from trout macrophage culture stimulated with lipopolysaccharide (LPS) and then passed through a Polymyxin B column to remove LPS. Results show that trout macrophage culture supernatants exhibit TNF-like activities. The supernatants significantly enhanced neutrophil migration and macrophage respiratory burst activity as assessed by NBT reduction test. The supernatants were also highly cytotoxic to murine L929 cells, which are known to be sensitive to mammalian TNFalpha. The biological activities of TNF in the trout macrophage culture supernatant was determined as 2.6 U ml(-1) in the presence of actinomycin D. This indicates biological cross-reactivity of trout TNFalpha-like factor on mammalian cells. Moreover, these activities were inhibited by a rabbit anti-mTNFalpha antibody. These results suggest that rainbow trout macrophages produce a TNFalpha-like factor that is similar to the mammalian TNFalpha in functions.     

Macrophage-derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS-stimulated macrophages

In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-alpha and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal(-)) and bound (D-gal(+)) fractions, with MNCF being found in the D-gal(+) fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal(-) fraction. Furthermore, the incubation of the D-gal(-) fraction with anti-TNF-alpha plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal(+) activity. Overall, these results suggest that the D-gal(-) inhibitory effect is partially mediated by TNF-alpha and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal(+) fraction.     

The macrophage-derived neutrophil chemotactic factor, MNCF: a lectin with TNF-alpha-like activities on neutrophils

The macrophage-derived neutrophil chemotactic factor (MNCF) is an α-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-α the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-α effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain.     

Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1

The aim of this study was to characterize the mediators released by mast cells responsible for IL-8-induced neutrophil migration. It was observed that IL-8 induces a dose-dependent neutrophil migration into peritoneal cavity of rats, but not into air-pouch cavity in which resident mast cells are not present. The transference of peritoneal mast cells to the air-pouch renders this cavity responsive to IL-8. The neutrophil migration induced by IL-8 into the peritoneal cavity was not observed when the peritoneal-resident mast cells were depleted by compound 48/80 or distilled water treatment. Confirming the importance of mast cells, IL-8-stimulated mast cells supernatant induced significant neutrophil migration when injected into peritoneal and air-pouch cavities. The IL-8-induced neutrophil migration was observed not to be dependent on LTB(4), prostaglandins or TNF-alpha, since MK886, indomethacin or thalidomide were unable to block the IL-8-induced neutrophil accumulation 'in vivo' or the release of neutrophil chemotactic factor "in vitro" by IL-8-stimulated mast cells. However, dexamethasone, an inhibitor of the synthesis of pro-inflammatory cytokines, blocked the neutrophil migration induced by IL-8 "in vivo" and also inhibited the release of the neutrophil chemotactic factor by IL-8-stimulated mast cells. Moreover, the incubation of IL-8-stimulated mast cells supernatant with antibody against cytokine-induced neutrophil chemoattractant 1 (CINC-1), but not against TNF-alpha or IL-1beta, inhibited its neutrophil chemotactic activity. Furthermore, we found a significant amount of CINC-1 in this supernatant. In conclusion, we demonstrated that the neutrophil migration induced by IL-8 is dependent on CINC-1 release from mast cells.     

Porcine spermadhesin PSP-I/PSP-II stimulates macrophages to release a neutrophil chemotactic substance: modulation by mast cells

The complex of porcine seminal plasma heterodimers I and II (PSP-I/PSP-II), which are heterodimers of glycosylated spermadhesins, is the major component of porcine seminal fluid. The proinflammatory and immunostimulatory activities of this spermadhesin complex suggest its participation in modulation of the uterine immune activity that may ensure reproductive success. Spermadhesin PSP-I/PSP-II induced the migration of neutrophils into the peritoneal cavity of rats via activation of resident cells. In the present study, we have investigated the involvement of macrophages and mast cells in the neutrophil chemotactic activity of PSP-I/PSP-II and the underlying mechanism. Macrophages and mast cells were isolated, cultured, and stimulated with purified PSP-I/PSP-II. Pharmacological modulation was performed using the glucocorticoid dexamethasone, indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and the supernatant of spermadhesin-stimulated mast cells. Macrophages stimulated with PSP-I/PSP-II released into the culture supernatant a neutrophil chemotactic substance. This activity was partly inhibited by both dexamethasone (85%) and the supernatant of spermadhesin-stimulated mast cells (74%) but not by indomethacin and MK886. An anti-tumor necrosis factor (TNF) alpha antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNFalpha by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.     

The macrophage-derived lectin, MNCF, activates neutrophil migration through a pertussis toxin-sensitive pathway.

The macrophage-derived neutrophil chemotactic factor (MNCF) is a D-galactose-binding lectin that induces neutrophil migration in vitro and in vivo. Neutrophil recruitment induced by MNCF is resistant to glucocorticoid treatment and is inhibited by the lectin-specific sugar, D-galactose. In the present study, we characterized the binding of MNCF to neutrophils and the responses triggered by this binding. Exposure to MNCF resulted in cell polarization, formation of a lamellipodium, and deep ruffles on the cell surface. By confocal microscopy, we observed that MNCF was evenly distributed on the cell surface after 30 min of incubation. The labeling intensity progressively diminished with longer incubations. Internalization kinetics showed that MNCF/ligand complexes were rapidly internalized, reaching maximum intracellular concentrations at 120 min and then decreased thereafter. The binding and internalization of MNCF were selectively inhibited by D-galactose. MNCF-induced neutrophil chemotaxis was inhibited by pertussis toxin. This fact strongly suggests that the MNCF-ligand on the neutrophil surface is a G-protein-coupled receptor (GPCR), similar to receptors for well-established neutrophil attractants. Our observations on the ability of MNCF to activate neutrophils are consistent with the increasing evidence for the participation of animal lectins in the innate immune response.     

A pre-formed Pyrogenic Factor Released by Lipopolysaccharide Stimulated Macrophages

The aim of this study was to investigate the pyrogenic activity of factor(s) released by rat peritoneal macrophages following a brief stimulation with LPS. The effect of this factor on the number of circulating leukocytes and serum Fe, Cu and Zn levels, was also evaluated. The possibility that the content of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF) in the supernatant could explain the observations was investigated. Supernatant produced over a period of 1 h by peritoneal macrophages, following a 30 min incubation with LPS at 37 degrees C, was ultrafiltered through a 10 000 MW cut-off Amicon membrane, sterilized, and concentrated 2.5, 5, 10 and 20 times. The intravenous (i.v.) injection of this supernatant induced a concentration-dependent fever in rats with a maximal response at 2 h. The pyrogenic activity was produced by macrophages elicited with thioglycollate and by resident cells. The supernatants also induced neutrophilia and reduction in Fe and Zn 6 h after the injection. Absence of activity in boiled supernatants, or supernatants from macrophages incubated at 4 degrees C with LPS, indicates that LPS was not responsible for the activity. In vitro treatment with indomethacin (Indo), dexamethasone (Dex), or cycloheximide (Chx) did not modify the release of pyrogenic activity into the supernatant or its effects on the reduction in serum metal levels. Although Chx abolished the production of mediator(s) inducing neutrophilia, and Dex reduced the induction of IL-1beta, TNF and IL-6, injection of the highest concentration of these cytokines detected in the supernatants did not induce fever. In vivo treatment with Dex, but not Indo, abolished the fever induced by the supernatant. These results suggest that macrophages contain pre-formed pyrogenic mediator(s), not related to IL-1beta, IL-6 or TNF, that acts indirectly and independently of prostaglandtn. It also seems likely that the pyrogenic activity is related to the factor responsible for the reduction of serum Fe and Zn levels, but not the neutrophilia.     

Nonsteroidal Anti-Inflammatory Drugs Increase Tumor Necrosis Factor Production in the Periphery but Not in the Central Nervous System in Mice and Rats

Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostaglandin (PG) synthesis, augment production of tumor necrosis factor (TNF) in most experimental models. We investigated the effect of two NSAIDs, indomethacin and ibuprofen, on the production of TNF in the CNS induced by intracerebroventricular injection of lipopolysaccharide (LPS). Indomethacin and ibuprofen, administered intraperitoneally, augmented (three- to ninefold) the levels of TNF in serum and peripheral organs of mice injected intraperitoneally with LPS and in rats with adjuvant arthritis (up to a sevenfold increase). However, NSAIDs (intraperitoneally or intracerebroventricularly) did not increase brain TNF production induced by intravenous LPS. In fact, indomethacin decreased (1.4–1.8-fold) TNF levels in the spinal cord of rats with experimental autoimmune encephalomyelitis and in the cortex of rats with focal cerebral ischemia. Systemic administration of iloprost inhibited serum TNF levels after intraperitoneal LPS, whereas intracerebroventricular injection of iloprost or PGE₂ did not inhibit brain TNF induced by intracerebroventricular LPS. Both peripheral and central TNF productions were inhibited by cyclic AMP level-elevating agents or dexamethasone. Thus, a PG-driven negative feedback controls TNF production in the periphery but not in the CNS.     

An intravascular chemoattractant lectin inhibits neutrophil migration

KM+, a lectin purified from Artocarpus integrifolia seeds, is an attractant for neutrophils, and has properties similar to fMLP, IL-8 and MNCF. The endogenous lectin MNCF, inhibits carrageenan-induced neutrophil migration when intravenously administered in rats. In an attempt to mimic the activity of MNCF with KM+, we determined the effect of intravenous (iv) injection of KM+ (5 g) on neutrophil migration to the peritoneal cavity of Wistar rats induced by KM+ (50 g, intraperitoneal, ip), fMLP (5 ng, ip) and carrageenan (300 g, ip). Initially we evaluated the effect of the time interval between intravenous and intraperitoneal administration of KM+. The intervals ranged from 20 to 120 min and progressively stronger inhibition was observed with increasing time intervals up to a maximum of 60 min, with effect decreasing thereafter. With injections at the optimum interval of 60 min, we observed that KM+ inhibited KM+- and carrageenan-induced neutrophil migration by 72%, and fMLP-induced migration by 56%. White cell counts for Wistar rats that only received KM+iv, performed at 0 to 120 min intervals after injection, revealed early neutropenia lasting 60 min, followed by a marked increase in circulating neutrophils that reached a maximum of twice the initial levels within 90 min and after 120 min returned to levels near to that observed before intravenous administration of KM+. These results indicate that when KM+ is present in the intravascular space, it produces an inhibitory effect on neutrophil migration similar to that caused by the intravenous administration of other chemoattractants, regardless of whether they act through a mechanism independent of carbohydrate recognition, as does IL-8, or are dependent on carbohydrate recognition, like MNCF.     

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.     

Nitric oxide inhibits neutrophil migration by a mechanism dependent on ICAM-1: role of soluble guanylate cyclase.

In the present study, we addressed the role of intercellular adhesion molecule type 1 (ICAM-1/CD54) in neutrophil migration to inflammatory site and whether the inhibitory effect of nitric oxide (NO) upon the neutrophil rolling, adhesion and migration involves down-modulation of ICAM-1 expression through a cyclic GMP (cGMP) dependent mechanism. It was observed that neutrophil migration induced by intraperitoneal administration of endotoxin (LPS), carrageenan (Cg) or N-formyl peptide (fMLP) in ICAM-1 deficient (ICAM-1-/-) is similar to that observed in wild type (WT) mice. The treatment of mice with NO synthase (NOS) inhibitors, NG-nitro-l-arginine, aminoguanidine or with a soluble guanylate cyclase (sGC) inhibitor, ODQ enhanced LPS- or Cg-induced neutrophil migration, rolling and adhesion on venular endothelium. These parameters induced by LPS were also enhanced by 1400 W, a specific iNOS inhibitor, treatment. On the other hand, the treatment of the mice with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, reduced these parameters induced by LPS or Cg by a mechanism sensitive to ODQ pretreatment. The NOS inhibitors did not enhance LPS-, Cg- or fMLP-induced migration and adhesion in ICAM-1-/- mice. Moreover, genetic (iNOS-/- mice) or pharmacological inhibition of NOS or of sGC enhanced LPS-induced ICAM-1 expression on mesenteric microcirculation vessels of WT mice. By contrast, SNAP reduced the ICAM-1 expression by a mechanism dependent on cGMP. In conclusion, the results suggest that although during inflammation, ICAM-1 does not contribute to neutrophil migration, it is necessary for the down-modulatory effect of inflammation-released NO on the adhesion and transmigration of neutrophils. Moreover, these NO effects are mediated via cGMP.     

Endogenous Tumor Necrosis Factor (TNF) α Mediates Neutrophil Accumulation at the Mid-Phase of a Murine Model of Pseudomonas aeruginosa Pneumonia

To determine the role of endogenous tumor necrosis factor (TNF) α on neutrophil influx into the lungs in acute Pseudomonas aeruginosa pneumonia, we evaluated TNF α activity, inflammatory cell response and neutrophil chemotactic activity in the bronchoalveolar lavage fluids (BALFs) of P. aeruginosa-infected mice. In the case of fatal pneumonia, the TNF α activity in the BALFs appeared within 3 hr, peaked at 6–12 hr and attenuated within 24 hr after intratracheal challenging, while no TNF α activity was detected in the plasma. The elevation of TNF α activity in the BALFs was closely associated with neutrophil accumulation. Mirroring the TNF α activity response and the influx of neutrophils into the murine airway, the number of neutrophils in the BALFs increased within 3 hr, peaked at 6–12 hr and remained elevated up to 24 hr after challenging. Neutralization of the TNF α activity in the BALFs with anti-murine TNF antiserum decreased the level of neutrophil migration by BALF 45.0–49.7% at 6 hr and 49.3–54.2% at 12 hr, while the neutralizing antiserum had no effect on the level of neutrophil migration by BALFs at 3 and 24 hr. Furthermore, the intravenous administration of anti-murine TNF antiserum 2 hr before challenging significantly inhibited neutrophil migration into the lungs of mice with sublethal pneumonia (P < 0.05; compared with mice receiving pre-immune serum). These data suggest that intra-alveolar TNF α plays an important role in causing lung neutrophil accumulation at the mid-phase of murine P. aeruginosa pneumonia.     

Comparative study of several lymphocyte functions in two strains of mice with different models of endotoxic shock

Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFalpha production were found to be associated with oxidative stress in endotoxin-induced septic shock. However, in this type of oxidative stress the lymphocyte involvement has rarely been studied. In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg). In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e. adherence to tissues and directed migration (chemotaxis), were studied. At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFalpha released by lymphocytes was also analyzed. The results show that endotoxic shock increases the adherence and TNFalpha release, and decreases the chemotaxis of peritoneal lymphocytes. These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock.     

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.     

Immunostimulating properties of synthetic derivatives of muramyl dipeptide and glucosaminyl muramyl dipeptide in vitro

Production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by macrophages of the spleen and peritoneal exudate of mice as well as cytotoxic factors (CFs) by murine splenocytes after in vitro activation was estimated. All the derivatives of muramyldipeptide (MDP) and glucosaminylmuramyldipeptide (GMDP) were able to induce production of TNF and CFs. In the presence of lipopolysaccharide (LPS), the effect was always higher. The response of the spleen macrophages to the effect of the preparations was higher than that of the peritoneal ones and ++non-fractionated splenocytes. GMDP and GMDP4 especially in the presence of LPS had the highest effect on induction of IL-1 by the murine peritoneal macrophages. On the contrary, MDP induced higher IL-1 synthesis by the spleen macrophages. The most active substances with respect to production of TNF, CFs and IL-1, i.e. MDP3 and GMDP4, might be recommended for immunotherapy of syngeneic tumors in animals.     

Inflammatory process caused by intraperitoneal injection of polyester in the rat: chemotactic activity in lavage fluid from the cavity

Minced polyester threads introduced into peritoneal cavity of guinea pigs or rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they may be exogenous and/or endogenous. These are released locally by the cells involved in inflammation. In this paper the chemotactic effects of the peritoneal fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The peritoneal cavity fluids were obtained by washing the cavity of untreated rats or rats intraperitoneally injected with polyester, 1, 3, 7, 14 days after the intraperitoneal injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear leukocytes from normal or treated rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrated that the peritoneal fluids taken 3 and 7 days after the intraperitoneal polyester injection, elicit an evident chemotaxis response greater than that showed by peritoneal fluids from control rats. It is suggested that chemotactic factors can be produced and released by mononuclear cells involved in the inflammatory process.     

Inhibitory effects of morphine on some inflammation-related parameters in the goldfish Carassius auratus L

Acute peritonitis induced in the goldfish by intraperitoneal injection of a sterile Thioglycollate solution shows a typical pattern with intraperitoneal exudation of serum proteins followed by influx of leucocytes (mainly heterophils/macrophages) correlated with elevated levels of chemotactic factors in peritoneal fluid and blood plasma. Supplementation of Thioglycollate with morphine (20 mg kg(-1) b.w.) does not affect the leakage of serum proteins into peritoneum. In contrast, it reduces the number of exudate peritoneal leucocytes (among them heterophils/macrophages) to the control level and decreases the level of peritoneal fluid/plasma chemoattractants, both effects being reversed by naltrexone pretreatment. Morphine itself acts as a chemokinetic factor for fish leucocytes as it increases their random movements. Therefore inhibitory effects of morphine on accumulation of exudate cells might be explained by inhibition of the production/release of chemotactic factors and/or reduced sensitivity of leucocytes to chemotactic signals. The effects of morphine on the goldfish peritonitis are in concordance with those described recently in Atlantic salmon and CB6 mice.