Effect of endothelins on human neutrophil activation by immune complexes.

Neutrophils are important effector cells of tissue injury in several pathological conditions, among them, immune complexes (IC)-induced inflammation and tissue injury. There is evidence that endothelins modulate IC-induced tissue injury in experimental models in vivo. In the present study we investigated the effect of endothelins on neutrophil activation by IC in vitro. To this purpose, pre-formed insoluble immune complexes were used to stimulate human neutrophils and production of leukotriene B(4) (LTB(4)) and hydrogen peroxyde (H(2)O(2)) were measured as indicative of phospholipase A(2) and oxidative burst activation and myeloperoxidase (MPO) release as indicative of cell degranulation. The effect of endothelins (ETs) in these events induced by IC was then examined. We found that IC stimulated all three events in human neutrophils. Addition of ET-1 but not ET-2 or ET-3 to the IC-stimulated neutrophils potentiated LTB(4) but not H(2)O(2) production. The endothelins added to resting neutrophils did not induce LTB(4) production but they were effective to stimulate H(2)O(2) production. The increased MPO activity induced by IC was not affected by endothelins nor did they stimulate the release of this enzyme in resting cells. These results show that endothelins are able to activate some neutrophil functions and to upregulate the IC-induced production of the pro-inflammatory molecule LTB(4). These data indicate that products of endothelial cells, such as endothelins, can be involved in the potentiation of neutrophil-dependent tissue injury.

Modulation of allergic and immune complex-induced lung inflammation by bradykinin receptor antagonists.

OBJECTIVE: The effect of bradykinin (B(1) or B(2)) receptor antagonists was studied in allergic and immune-complex-induced lung inflammation. METHODS: Lungs of BALB/c mice were examined 24 h after induction of lung inflammation, either allergic (ovalbumin-sensitized submitted to two aerosol of antigen, one week apart) or immune-complex induced (intratracheal instillation of IgG antibodies followed by intravenous antigen). The bradykinin B(2) receptor antagonist, HOE-140 or bradykinin B(1) receptor antagonist, R-954 were given intraperitoneally (100 microg/kg), 30 min before induction. RESULTS: In allergic inflammation, pre-treatment with R-954 reduced eosinophil infiltration into the lungs, mucus secretion and the airway hyperreactivity to methacholine. Pre-treatment with HOE-140 increased eosinophil infiltration but did not affect the other parameters. In immune-complex inflammation, HOE-140 increased neutrophil infiltration but not their activation nor the hemorrhagic lesions. R-594 pre-treatment did not change the parameters examined. CONCLUSION: These results show important modulatory effects of bradykinin B(1) and B(2) receptor antagonists in both models of lung inflammation.

Platelet-activating factor and eicosanoids are mediators of local and systemic changes induced by immune-complexes in mice.

A passive Arthus reaction (AR) induced in the peritoneal cavity of mice was followed by increased local vascular permeability and haemoconcentration. The intensity of the increased vasopermeability was higher in BALB/c compared with C3H/HePas mice despite the latter being 10 times more sensitive to platelet-activating factor (PAF). C3H/HePas mice however, exhibited higher levels of haemoconcentration and shock-like symptoms. Both events were inhibited by the PAF antagonist, WEB 2170. Indomethacin reduced both pathological events whereas L663,536, that inhibits leukotrienes synthesis reduced haemoconcentration but only in BALB/c mice. PAF was released into the peritoneal cavity, peak release being at 10 min after induction of AR. Prostaglandin E2 (PGE2), thromboxane B2 (TXB2), leukotriene B4 (LTB4), and leukotriene C4/D4 (LTC4/D4) were also released at this time. Similar levels of PAF and eicosanoids were found in BALB/c and C3H/HePas mice except for LTB4, which was higher in C3H/HePas. It is concluded that PAF and eicosanoids are mediators of local and systemic changes induced by immune complexes in the peritoneal cavity of mice.

Modulation by lipid mediators of immune complex-induced lung inflammation in mice.

The present study characterized a murine model of immune complex-induced pneumonitis and investigated the role of platelet-activating factor (PAF) and eicosanoids as mediators of lung neutrophil infiltration and hemorrhagic lesions. Rabbit antibodies to bovine serum albumin were injected into the airways and bovine serum albumin was injected intravenously into C3H/HePas and BALB/c mice. After 24 h, a significant increase in neutrophil infiltration and hemoglobin concentration in the bronchoalveolar lavage fluid and lung parenchyma was observed in both strains despite the C3H/HePas strain being 10 times more sensitive to PAF. Neutrophil influx and vascular lesions were not affected by pre-treatment of the mice with the PAF receptor antagonist, WEB 2170 (5-(2-chlorphenyl)carbonyl)-3,4-dihydro- 10-methyl-3-((4-morpholinyl)-2H,7H-cyclopenta(4,5)thieno(3,2-f)(1,2,4)-t riazolo-(4,3-a)(1,4)-diazepine). In contrast, neutrophil influx and vascular lesions were increased by the cyclo-oxygenase inhibitor, indomethacin, and reduced by the inhibitor of leukotriene synthesis, MK 886 (3-[1-(4-chlorobenzyl-3-t-butyl-thio-t-isopropyl-indol-2y-1]-2-2-+ ++dimethylpropanoic acid) and by the leukotriene B4 receptor antagonist, RO 0254094 (2-[(5-carboxypentyl)-6-[6-[3,4-dihidro-4-oxo-8-propyl-2H-1-benzop yran-7-yl)hexyl] benzenepropanoic acid). Increased levels of leukotriene B4, leukotriene C4/D4, thromboxane B2 were found in bronchoalveolar lavage fluid 4 h after induction of the reaction. There is also a tendency to increased prostaglandins E2 levels. Neutrophil infiltration and vascular lesions in immune complex-induced pneumonitis in mice are mediated by leukotriene B4.

Lipid mediators, tumor necrosis factor and nitric oxide and their interactions in immune-complex-induced lung injury.

We investigated the contribution of eicosanoids, platelet-activating factor, tumor necrosis factor and nitric oxide to the neutrophil influx and development of pulmonary haemorrhagic lesions following immune-complex-induced pneumonitis in rats and possible interactions between these mediators. Increased levels of leukotriene B4 and tumor necrosis factor, measured by enzyme immunoassay and L-929 cytotoxicity assay, were found in the bronchoalveolar lavage 1 and 4 h after induction of the reaction, respectively, and their release was dependent on the previous generation of platelet activating factor. Antagonism of leukotriene B4 receptors by RO-0254094 (2-[(5-carboxypentyl])oxy]-6-[6-[3,4-dihydro-4-oxo-8-propyl-2H-1-benzopy ran-7-yl)oxy]hexyl] benzenepropanoic acid), inhibition of nitric oxide synthesis by L-NAME (Nw-nitro-L-arginine methyl ester) and antagonism of PAF-receptors by WEB-2170 (5-(2-chlorphenyl)-3-4-dihydro-10-methyl-3-((4-morpholinyl)carbony l)-2 H,7H-cyclopenta (4,5)thieno(3,2-f)(1,2,4)-triazolo-4,3,a)91,4)diazepine), significantly inhibited the intensity of haemorrhage, evaluated by the increased levels of extravascular hemoglobin in homogenates of lung tissues. Little evidence support the role of tumor necrosis factor in these lesions. The infiltration of neutrophils, evaluated by measuring myeloperoxidase in homogenates of lungs, was reduced by compounds L-663,536 (3-[1-(4 chlorobenzyl)-3-t-butyl thio-5-isopropylindol-2-yl]-2-2-dimethylpropanoic acid), WEB-2170 and L-NAME. These results indicate that neutrophil infiltration and haemorrhagic lesions in immune-complex-induced lung inflammation are mediated by platelet activating factor, leukotriene B4 and nitric oxide and point out to interesting interactions between these mediators.

Roles of endothelins and their receptors in immune complex-induced/polymorphonuclear-mediated lung injury (reversed passive arthus reaction) in CD-1 mice.

A number of pro-inflammatory mediators (leukotrienes, platelet activating factor, cytokines) participate in the process of neutrophil-dependent lung injury induced by immune complexes. Here, we studied the role of endothelins (ET) in the reversed passive Arthus reaction (AR) as a model of pneumonitis in CD-1 mice. We examined the broncholaveolar lavage fluid (BALF) for signs of inflammation such as the accumulation of cells, myeloperoxidase (MPO) activity and hemoglobin (Hb) levels, as a measure of hemorrhagic lesions, 24 h after injection. We used a selective ETA (BQ-123) or a non-selective ETA/ETB-R (SB 209670) receptor antagonist at various concentrations (2.5, 5 or 10 mg/kg ip at -8, 0, 8 and 16 h) to assess the involvement of ET. Challenged mice revealed signs of acute inflammation and hemorrhagic lesions. Levels of Hb and MPO, total and neutrophil cell counts increased by 9-, 9-, 3.2- and 63-fold, respectively. The lower dose of SB 209670 reduced Hb levels by 21% (P<0.05), without affecting cell accumulation or MPO. The mid-dose had no effect; the highest dose caused 60, 57 and 70% increases in Hb levels, total cell and neutrophil counts, respectively. Conversely, the highest dose of BQ-123 decreased Hb, total cell and neutrophil counts and MPO levels by 36, 35, 42 and 70%, respectively. These results support a role for ET in AR lung injuries. They also suggests that blocking ETA-R may be beneficial, while blockade of ETB-R (using a high dose of SB 209670) may be detrimental. A beneficial ETB-mediated response may exist that naturally interferes with events triggered by the formation of immune complexes such as cell accumulation and their subsequent activation leading to acute lung injury.

A new murine model of pulmonary eosinophilic hypersensitivity: contribution to experimental asthma.

BACKGROUND: We have recently described a model of hypersensitivity reaction in the mouse paw, which induces a typical late-phase reaction with a marked eosinophilic infiltrate. OBJECTIVE: In the search for a murine model of asthma, this model was adapted to the lungs and compared with other models of pulmonary hypersensitivity. METHODS: A fragment of heat-coagulated hen's egg white was implanted subcutaneously, and 14 days later, the mice were challenged intratracheally with aggregated ovalbumin. Comparison was made with a group that received subcutaneous injection of soluble ovalbumin in alumen, challenged as described above and with four additional protocols of immunization and challenge. RESULTS: Forty-eight hours after challenge, the percentage of eosinophils was higher in the egg white implant group (35%) than in the group immunized with ovalbumin in alumen (10.4%). The eosinophil peroxidase activity in lung homogenates of the first group was also significantly higher (529 ng/ml) than that of the second group (43 ng/ml). These results were reproduced in five different mouse strains. Compared with five different models of lung hypersensitivity, the egg white implant model was unique in terms of persistence of the pulmonary eosinophilia. Histopathologic analysis of the lungs of mice immunized with egg white implant showed peribronchial, perivascular, and intraepithelial eosinophil infiltration; morphologic characteristics of bronchoconstriction; and patchy epithelial shedding. At 21 days, in addition to persistence of eosinophil infiltrate, enlarged alveoli, reflecting air trapping, were observed. CONCLUSION: On the basis of the characteristics of the model described here, we propose it as a suitable murine model of asthma.

Platelet-activating factor: the effector of protein-rich plasma extravasation and nitric oxide synthase induction in rat immune complex peritonitis.

1. The involvement of platelet-activating factor (PAF) in immune complex-induced/polymorphonuclear-mediated tissue injury was studied by use of a reverse passive Arthus (RPA) model in the peritoneal cavity of rats. 2. Extravasation of protein-rich plasma, accumulation of polymorphonuclear leukocytes (PMN), and the production of nitric oxide (NO) by resident peritoneal mononuclear phagocytes were assayed. 3. Treatment of rats with either UR-12460 or BB-823, two compounds which possess different chemical structures, but elicit the same antagonistic effect on the PAF receptor, abrogated protein-rich plasma extravasation. In contrast, they did not show any effect on the accumulation of PMN. 4. Inhibition of NO production with both NG-mono methyl-L-arginine and NG-nitro-L-arginine failed to prevent protein-rich plasma extravasation. 5. The production of NO by peritoneal adherent cells following RPA was measured in cells maintained for 2 to 28 h in culture, and it was significantly increased in cells removed as early as 15 min after RPA induction, as compared to controls. 6. Addition of 10 nM PAF to the culture medium reduced the generation of NO by peritoneal cells from RPA rats, whereas this mediator enhanced NO production in cells from naive control animals. 7. Treatment with either UR-12460 or BB-823 prior to the induction of RPA produced an almost complete inhibition of NO production. 8. Assay of nitric oxide synthase activity in cell homogenates from peritoneal cells showed that the activity was due to the inducible form of the enzyme. 9. Study by Northen blotting of mRNA coding for the inducible NO synthase (iNOS) showed transcription at 6 and 18 h after the induction of RPA, which was inhibited in UR-12460-treated rats.10. These data indicate that PAF is the main mediator of the early plasma leakage observed in RPA,and also that PAF is implicated in the triggering of long-term changes via induction of specific genes, as judged from its ability to promote the expression of iNOS.

Inhibition by insoluble immune complexes of both capacitative Ca2+ entry and Ca2+ mobilization by chemotactic agonists in human neutrophils.

Insoluble immune complexes (IIC) stimulate human neutrophils by binding to their FcR. It is known that they are able to release Ca2+ from intracellular stores but they induce little Ca2+ entry from the extracellular medium, a dissociation that cannot be explained within the framework of the capacitative model for Ca2+ entry, which is well established for these cells. We show here that IIC induce a strong and long-lasting inhibition of the Ca2+ pathway activated by emptying the Ca2+ stores (capacitative Ca2+ entry), which develops simultaneously with the activation of Ca2+ release from intracellular stores. This inhibition strongly resembles that previously described effected by FMLP and by phorbol dibutyrate, which seems to be mediated by phosphorylation. Inhibition by IIC, however, differs from that induced by FMLP and phorboldibutyrate in its lack of sensitivity to cytosolic-free calcium concentration and in its different sensitivity to the protein kinase inhibitors staurosporin and chelerythrine. It was also insensitive to the protein tyrosine kinase inhibitors genistein and herbimycin A. We also show that IIC inhibit Ca2+ mobilization induced by other agonists and that this inhibition is potentiated by the protein phosphatase inhibitor calyculin A. Our results therefore suggest that binding of IIC to the FcR activates a protein phosphorylation mechanism leading to a long-lasting inhibition of both capacitative Ca2+ entry and Ca2+ mobilization induced by other agonists such as FMLP, platelet-activating factor, or leukotriene B4.

Study of the effector mechanism involved in the production of haemorrhagic necrosis of the small intestine in rat passive anaphylaxis.

1. The effector mechanism of intestinal necrosis in rat anaphylaxis was studied following several complementary approaches: (i) the use of monoclonal antibodies (mAb) belonging to different classes (IgG1, IgG2b and IgE anti-DNP), (ii) the assay of mediators, and (iii) the use of pharmacological tools. 2. Lethality and haemorrhagic necrosis of the small intestine were observed in IgE-sensitized rats, whereas IgG mAb produced milder physiological disturbances. 3. Inhibition of leukotriene biosynthesis reduced the drop of systemic blood pressure (BP) and the extent of protein-rich plasma exudation but it did not influence the haemorrhagic component of intestinal necrosis. 4. The antihistamine, pyrilamine, partially diminished the haemorrhagic component of the intestinal necrosis. 5. The involvement of mediators related to platelet-activating factor (PAF) was studied by examining the pharmacological effects of these autacoids and of PAF-receptor antagonists (PCA4248, UR12460 and BB823). PAF induced intestinal lesions similar to those observed in IgE-sensitized rats and PAF-receptor antagonists markedly decreased haemorrhage in IgE-sensitized rats. 6. PAF levels were transiently increased after dinitrophenol (DNP)- bovine serum albumin (BSA) challenge in the small intestine of IgE-sensitized rats. 7. These data stress differences in the outcome of anaphylaxis related to the type of receptors for the Fc portion of immunoglobulins that are involved. IgE is the antibody class that elicits the most severe response due to the activation of mast cells via Fc epsilon RI (surface receptors that bind IgE antibodies with high affinity), and the only one able to produce intestinal haemorrhagic necrosis. 8. The mast-cell-derived mediators PAF/acyl-PAF and histamine, most probably associated with tumour necrosis factor alpha/cachectin (TNF-alpha), seem to play a central role in the production of the vascular changes required for the extravasation of erythrocytes in the small intestine mucosa.

Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila.

The blood cells of the pulmonate snail Biomphalaria tenagophila, an important transmitter of the trematode Schistosoma mansoni in Brazil, were examined by light and transmission electron microscopy (TEM). Two hemocyte types were identified: hyalinocytes and granulocytes. Hyalinocytes are small young (immature), poorly spreading cells, which have a high nucleocytoplasmic ratio and are especially rich in free ribosomes. They do not appear to contain lysosome-like bodies and represent less than 10% of the circulating hemocytes. Granulocytes are larger hemocytes which readily spread on glass surface and which strongly react to the Gomori substrate, indicating the enzyme acid phosphatase usually found in lysosomes. Ultra-structurally, they contain a well-developed rough endoplasmic reticulum, dictyosomes and some lysosome-like dense bodies. Granulocytes do not exhibit a characteristic granular aspect and the few granules observed in the cytoplasm should correspond to a lysosome system. They were named granulocytes instead of amoebocytes to use the same terminology adopted for Biomphalaria glabrata in order to make easier comparative studies. This is a preface study for more specific investigations on the functional activities of the blood cells of B. tenagophila and their interactions with the trematode parasite.