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.     

Polyester treatment in rats with a dorsal air pouch: chemotaxis in lavage fluid from the pouch]

Minced polyester threads introduced into peritoneal cavity of rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they are released locally by cells involved in inflammation. In this paper the chemotactic effect of the peritoneal and subcutaneous air pouch fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The fluids were obtained by washing the cavity of untreated rats or rats injected with polyester, 7 days after the injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear cells from normal rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrate that a chemotactic activity is present in peritoneal and subcutaneous air pouch fluids following the inflammatory process. In conclusion the chronic inflammation determines the appearance of chemotactic factors for PMN cells, in the peritoneal cavity and in the air pouch, and the air pouch is a very convenient experimental system with the particular advantage that it permits easy repeated sampling of exudate during the course of an inflammatory response.     

Differential regulation of chemokine production in human peritoneal mesothelial cells: IFN-gamma controls neutrophil migration across the mesothelium in vitro and in vivo.

Leukocyte recruitment into the infected peritoneal cavity consists of an early, predominant polymorphonuclear leukocyte (PMN) influx and subsequent, prolonged mononuclear cell migration phase. Although chemokine secretion by resident peritoneal cells plays a primary role in mediating this migration, the mechanisms involved in controlling the switch in phenotype of cell infiltrate remain unclear. The present study investigates a potential role for the Th1-type cytokine IFN-gamma in the process of leukocyte recruitment into the peritoneal cavity. Stimulation of cultured human peritoneal mesothelial cells with IFN-gamma (1-100 U/ml) alone or in combination with IL-1beta (100 pg/ml) or TNF-alpha (1000 pg/ml) resulted in significant up-regulation of monocyte chemoattractant protein-1 and RANTES protein secretion. In contrast, IFN-gamma inhibited basal and IL-1beta-, and TNF-alpha-induced production of IL-8. The modulating effects of IFN-gamma on chemokine production occurred at the level of gene expression, and the degree of regulation observed was dependent on the doses of IL-1beta and TNF-alpha used. Analysis of the functional effects of IFN-gamma on IL-1beta-induced transmesothelial PMN migration with an in vitro human transmigration system and an in vivo murine model of peritoneal inflammation demonstrated that IFN-gamma was able to down-regulate PMN migration induced by optimal doses of IL-1beta. These effects were mediated in vivo via down-regulation of CXC chemokine synthesis. These findings suggest that IFN-gamma may play a role in controlling the phenotype of infiltrating leukocyte during the course of an inflammatory response, in part via regulation of resident cell chemokine synthesis.     

Inflammatory reaction to polyester material in the guinea pig. V. Semiautomatic determination of peritoneal cell volume

The volume of the peritoneal cells of guinea pigs treated with injections of 1) minced polyester threads (Mersilene), 2) saline, and of untreated animals, has been determined utilizing a Coulter Counter coupled with a pulse height analyzer. The volumetric test on the whole cell population has emphasized a distinctly bimodal distribution, due to the presence of two cellular types picked out as first peak (P 1 degrees) and second peak (P 2 degrees), while the non-adherent cells (CNA) have shown an unimodal distribution. Statistically significant difference in the mean volumes have been found between the P 1 degrees and P 2 degrees cells and between the CNA and P 1 degrees cells. The percentage of CNA in the whole cell population significantly decreases in "Mersilene"-treated guinea pigs.     

The role of fractions from Paracoccidioides brasiliensis in the genesis of inflammatory response

The influx of inflammatory cells towards the peritoneal cavity in rats inoculated intraperitoneally with subcellular preparations of the fungus Paracoccidioides brasiliensis was studied. In addition to the dead fungus, also fractions F1 of the cell wall, which mainly consisted of polysaccharides and the lipid extract, induced intense cell migration 4 hr after inoculation, with a greatly increased number of polymorphonuclear leucocytes (PMN). Study of the kinetics of cell influx showed that both fraction F1 and the lipid extract initially induced intense PMN migration between the 4th and 24th hr after inoculation of these agents, followed by migration of mononuclear cells (MN) around the 48th hr. We also observed that migration of these cells increased gradually after inoculation of growing doses of fraction F1. The present data suggest that polysaccharides and lipids isolated from P. brasiliensis may participate in the initial phase of the inflammatory response in paracoccidioidomycosis.     

Nonpurulent response to toxic shock syndrome toxin 1-producing Staphylococcus aureus. Relationship to toxin-stimulated production of tumor necrosis factor

Infection of surgical wounds with toxic shock syndrome toxin 1 (TSST-1)-producing Staphylococcus aureus does not usually elicit a purulent response from the host. Because S. aureus is normally a pyogenic pathogen, this phenomenon suggests that strains of staphylococci that produce the exotoxin are able to inhibit the migration of polymorphonuclear neutrophils (PMN) to sites of infection. We have considered that inhibition of leukocyte migration may be an effect of secreted TSST-1 and have studied direct and indirect effects of the exotoxin on migratory functions of PMN in vitro. Preincubation of PMN with TSST-1 produced no inhibition of random motility or FMLP- or C5a-stimulated chemotaxis under agarose. Supernatant fluids from mononuclear leukocytes incubated with TSST-1, however, were potently inhibitory for both PMN random and chemotactic migratory functions. The inhibitor of migration was identified as TNF based upon neutralization by anti-TNF antiserum and its presence in the culture supernatant fluids assayed in terms of cytotoxicity for murine TNF-sensitive L-929 cell line cells. Preincubation of PMN with recombinant human TNF also inhibited subsequent PMN random and chemotactic migratory functions. We propose that TSST-1 inhibits the mobilization of PMN to sites of infection by stimulation of monocyte/macrophage TNF production and suggest that TNF may also contribute to some other effects of toxic shock syndrome.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Interference of polyester inflammation on the development of Yoshida ascites hepatoma in the rat

Minced polyester threads introduced into the peritoneal cavity cause a chronic inflammation with evidence of macrophage and lymphocyte stimulation. In this paper an interference between this kind of inflammation and the growth of Yoshida ascites hepatoma has been shown, which has been found to dependent on the time interval elapsed between the introduction of polyester (Mersilene) minces and injection of the hepatoma cells. Rats were treated intraperitoneally with Mersilene and then divided in to three groups: the first was injected intraperitoneally with hepatoma cells immediately (TM 0), the second after 7 (TM 7) and the third after 14 days (TM 14); rats untreated with polyester and implanted with the same number of hepatoma cells served as controls (NT). While in groups NT and TM 0 a rapid growth of hepatoma cells occurred, together with the accumulation of a considerable volume of ascitic fluid, no tumor growth neither ascite production occurred in groups TM 7 and TM 14; in these animals where several days were allowed to elapse after polyester introduction, the hepatoma cells which had been injected rapidly disappeared and were no more found 48 h after the intraperitoneal injection. It is suggested that the inhibition of the neoplastic growth may be dependent on the activation of macrophages (and possibly NK cells) which accompanies the development of the chronic polyester inflammation.     

Interleukin-4 is chemotactic for mouse macrophages.

An important component of the cell-mediated immune response often is the migration of macrophages to the site of immune activity. Although much evidence suggests that macrophage migration is regulated by antigen-specific T cells, the influence of T cell-derived cytokines on macrophage chemotaxis has not been well studied. Here we present evidence that interleukin-4 (IL-4), a cytokine derived from T helper 2 (Th 2) cells, is chemotactic for mouse peritoneal macrophages. In an in vitro chemotaxis assay using Boyden chambers, recombinant IL-4 was chemotactic for mouse peritoneal exudate macrophages. This response was inhibited in a dose-dependent manner by the anti-IL-4 antibody, 11B11. As shown here and previously, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), cytokines derived from T helper 1 cells, are not chemotactic for mouse macrophages.     

Clinical and pathological questions concerning the tumor-like giant cell granulomas of the peritoneal cavity

Seven patients with tumor-like granulomatous lesions of the peritoneal cavity were cured, except one, by correct surgical intervention removing the inflammatory hyperplastic tissues and restoring the permeability of the alimentary tract. The exact diagnosis was suggested by the existence in the personal history of the patients of one or several interventions on the peritoneal cavity (6 of 7), and was confirmed by intraoperative, sometimes repeated, microscopic examination, rendering evident a fibrogenous giant cell granulomatous process; the presence of foreign bodies, especially suture threads or crystals (the latter characterized in polarized light) is very helpful for the diagnosis of these tumor-like inflammations.     

Chemotactic response of splenic mononuclear cells to angiotensin II in murine schistosomiasis

Murine Schistosomiasis mansoni is a parasitic infection associated with a delayed-type hypersensitivity granulomatous reaction to the schistosome eggs. This reaction is characterized by the accumulation of mononuclear cells and other inflammatory cell types around the eggs. Granuloma macrophages produce angiotensin II (AII), which appears to have immunoregulatory function. By using an in vitro chemotaxis assay, this study demonstrated that AII is a chemotactic factor for splenic mononuclear cells derived from infected mice. The response was bimodal, with peak activities occurring at 10(-10) and 10(-6) M. AII was chemotactic for T lymphocytes, B lymphocytes, and a large population of unidentified mononuclear cells at the optimal chemotactic concentrations of the peptide. At high concentrations, AII was also chemotactic for phagocytic mononuclear cells. Sar1, ala8-AII, an analog of AII with antagonist activity, completely blocked AII-induced chemotaxis. A [3H]-AII binding assay revealed high-affinity specific binding on spleen cells. The binding was rapid, was dependent on radioligand concentration, and was reversible. These latter observations suggest that the chemotactic activity of AII for subpopulations of splenic mononuclear cells is mediated via AII receptors.     

Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse. Prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor

The i.p. injection of mice with highly purified recombinant human rIL-1 alpha or beta resulted in the rapid influx of a large number of polymorphonuclear neutrophils (PMN) into the peritoneal cavity. Significant increases in the number of PMN were induced by doses of IL-1 which ranged from 0.005 to 5 ng/injection. Interestingly the dose response for PMN influx was bell-shaped because 50 ng of IL-1 did not result in a significant increase in peritoneal PMN. IL-1 induced PMN infiltration was detectable by 1 h with peak levels of PMN obtained by about 2 h, followed by a subsequent decline by 24 h. Other cytokines, IL-2, IFN-gamma, IFN alpha beta, granulocyte-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, and TNF-beta were compared to IL-1 for their ability to induce a PMN influx into the peritoneum. Only TNF-alpha or TNF-beta (lymphotoxin) were able to induce a significant influx of PMN within 2 h. However, based on total protein administered, about 100 times more TNF than IL-1 was required to produce a comparable PMN infiltration. Intraperitoneal injection of inhibitors of the cyclooxygenase or lipoxygenase pathways did not inhibit the IL-1-induced influx of PMN. Also, neither IL-1 nor TNF triggered an increase in PG or leukotriene release from peritoneal cells in vitro. Furthermore, direct peritoneal injection of leukotriene B4, a potent PMN chemoattractant in vitro, did not induce any significant increase in PMN in the peritoneal cavity indicating that chemotactic activity alone is insufficient for inducing peritoneal infiltration. These results suggest that the local production of very low levels of IL-1 in vivo would be sufficient to initiate a sequence of events that results in a rapid accumulation of PMN. Because IL-1 was not chemotactic for PMN in vitro, our data suggest that IL-1 induces production of factors that are chemotactic for PMN. Alternatively, IL-1 may act on other stages of the complex sequence of events that regulates the emigration of PMN into tissue sites in vivo. The synergy apparent in PMN influx when suboptimal concentrations of IL-1 and TNF were injected suggests that the local production of very low concentrations of these cytokines in situ could play a critical role in the emigration of PMN during infection.     

Eosinophils and immune mechanisms: V. Demonstration of mouse spleen cell-derived chemotactic activities for eosinophils and mononuclear cells and comparisons with eosinophil stimulation promoter

The chemotactic response of mouse eosinophil-rich peritoneal exudative cells to the lymphokine eosinophil stimulation promoter (ESP) was examined. Both eosinophils and monocytes are chemotactically attracted across a 3-μm-pore size polycarbonate filter toward a concentration gradient of ESP-containing culture supernatant fluids. Deactivation of both cell types occurs following preincubation of the responding cells in culture supernates containing ESP activity. The chemotactic activity for both eosinophils and mononuclear cells is stable when incubated at 60 °C for 30 min but is labile at 80 °C, is nondialyzable, and at peak activity exhibits an apparent molecular weight of approximately 25,700 daltons, based on Sephadex G-100 gel chromatography. Production conditions required for the generation of chemotactic and ESP activities are identical, and fractions of culture supernatant fluids possessing one activity are also positive for the other. Preliminary results therefore indicate that the lymphokine ESP attracts both eosinophils and monocytes in a gradient-induced chemotaxis assay.     

The combined action of hyaluronic acid and fibronectin stimulates neutrophil migration

Previous investigations have demonstrated that the chemotactic response of polymorphonuclear leukocytes (PMN) was stimulated by hyaluronic acid (HA) when serum was present. The aim of the present investigation was to identify the serum factor necessary for the stimulation of PMN chemotaxis by HA. By means of gel filtration, the m.w. of the serum component was shown to be greater than 350,000. Immunoprecipitation of serum with anti-fibronectin, but not with anti-IgM and anti-alpha 2-macroglobulin, inhibited the stimulation of PMN chemotaxis by HA. Preincubation of PMN with HA (10 to 500 micrograms/L) and isolated fibronectin (0.1 to 100 mg/L) significantly stimulated the chemotactic response of PMN. Also, random migration of PMN was significantly increased by preincubation of the cells with HA (10 to 500 micrograms/L) and isolated fibronectin (50 to 200 mg/L). Additionally, PMN preincubated with HA (10 to 50 micrograms/L) and with fibronectin (10 to 50 mg/L) added afterwards, and PMN preincubated with fibronectin (10 mg/L) and with HA (5 to 10 micrograms/L) added after the preincubation, showed a significant stimulation of the chemotactic response. PMN preincubated with serum and chondroitin sulfate, or with fibrinogen and HA, demonstrated no stimulation of the chemotactic response. The present investigation suggests that the combined action of HA and fibronectin, which probably takes place at the cellular membrane, is a major mechanism in the HA-mediated stimulation of PMN migration.     

Production of C5a antagonist by synovial and peritoneal tissue fibroblasts

Fibroblasts grown from synovial and peritoneal tissues release into the medium an inhibitor of neutrophil chemotaxis. The inhibitor resembles the antagonist previously described in synovial and peritoneal fluids. It is a heat stable (56 degrees C) protein of MW approximately 40 kDa that counteracts the chemotactic activity of zymosan-activated serum or purified C5a but not the peptide chemoattractant F-met-leu-phe. No chemotactic inhibitor was detected in media from skin fibroblast cultures or in formal human sera. It is suggested that the inhibitor is produced locally by synovial and peritoneal fibroblasts and that it might play a role in the regulation of inflammation at sites lined with mesothelium.     

Staphylococcal enterotoxin B induced release of macrophage migration inhibition factor from normal lymphocytes

Staphylococcal enterotoxin B (SEB), a potent lymphocyte mitogen, inhibits migration of peritoneal exudate cells from most guinea pigs but does not inhibit migration of purified macrophages. Experiments were designed to test the ability of highly purified SEB to induce normal lymphocytes to release migration inhibition factor (MIF). Supernatants of lymph node lymphocytes cultured with SEB inhibited the migration of purified macrophages, indicating the release of a migration inhibition factor. Mitomycin-C blocked the SEB-induced release of MIF. SEB-induced MIF localized in the albumin fraction on Sephadex G-200 chromatography. Antibody to SEB specifically blocked the inhibitory effect of SEB on migration of normal guinea pig peritoneal exudate cells.     

Amphotericin B alters the affinity and functional activity of the oligopeptide chemotactic factor receptor on human polymorphonuclear leukocytes

Leukocyte chemotaxis is initiated by the binding of chemotactic factors to specific, high-affinity receptors. Amphotericin B, a polyene antibiotic that binds to membrane cholesterol, inhibits human neutrophil (PMN) chemotaxis. We examined the effects of this drug on PMN functions mediated by the oligopeptide chemotactic factor receptor. The antibiotic irreversibly inhibited chemotaxis and depressed the binding of the radiolabeled chemoattractant, fMet-Leu-[3H]Phe, to its receptor without affecting the receptor's specificity. The drug lowered the binding affinity of the receptor by up to fivefold and slightly increased its number. Doses of amphotericin B that depressed receptor affinity and inhibited chemotaxis did not diminish lysosomal enzyme secretion or superoxide anion production. Nystatin, a less potent polyene antibiotic, also diminished chemotactic factor binding, but to a lesser degree than amphotericin B did. A chemically unrelated antifungal agent had no effect on either binding or chemotaxis. Thus, pharmacologic manipulation can alter the affinity of the chemotactic factor receptor on human PMN; this alteration is associated with a change in receptor function. The data suggest that receptor affinity regulates or at least reflects its functional state, and that the transduction mechanisms for various biologic responses mediated by the chemoattractant receptor are heterogeneous. By pharmacologic alterations of receptor affinity, one may be able to modulate specific biologic responses elicited by chemoattractant receptor-ligand interactions.     

Inhibition of human neutrophil chemotaxis by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine

The protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine (C-I), inhibits superoxide release from human neutrophils (PMN) stimulated with phorbol myristate acetate or synthetic diacylglycerol, without inhibiting superoxide release from PMN stimulated with the chemoattractants C5a or N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). In this study, we investigated the effect of C-I on human PMN chemotaxis to C5a, f-Met-Leu-Phe, leukotriene B4 (LTB4), and fluoresceinated N-formyl-methionyl-leucyl-phenylalanine-lysine (f-Met-Leu-Phe-Lys-FITC). PMN, preincubated for 5 min at 37 degrees C with 0 to 200 microM C-I, were tested for their migratory responses to the chemoattractants. C-I (greater than or equal to 1 microM) significantly inhibited PMN chemotaxis to f-Met-Leu-Phe, f-Met-Leu-Phe-Lys-FITC, and C5a without affecting random migration. Maximal inhibition of chemotaxis to these attractants occurred with greater than or equal to 50 microM C-I, at which chemotaxis was inhibited by 80 to 95%. The C-I inhibition was reversible. In contrast, 200 microM C-I did not inhibit the number of PMN migrating to LTB4, although, the leading front of PMN migration to LTB4 was inhibited by C-I. C-I inhibited PMN orientation to C5a and f-Met-Leu-Phe without affecting orientation to LTB4. C-I did not inhibit the binding of radiolabeled f-Met-Leu-Phe or f-Met-Leu-Phe-Lys-FITC to PMN. These findings suggest that the chemotactic responses of PMN to f-Met-Leu-Phe and C5a involve a protein kinase-dependent reaction which is inhibited by C-I.     

Dexamethasone inhibits generation in inflammatory sites of the chemotactic activity attributable to leukotriene B4

Effects of dexamethasone on chemotactic activity to polymorphonuclear leukocytes (PMN) of a lipophilic fraction collected with the aid of octadecylsilyl silica cartridge from exudates of an allergic inflammation were investigated. The chemotactic activity of this fraction was attributable to leukotriene B4 fraction separated by means of a reversed phase high performance liquid chromatography. Local application of dexamethasone suppressed dose-dependently the chemotactic activity of the lipophilic fraction in parallel with the inhibition of PMN infiltration in the inflammatory sites.     

Role of 5-lipoxygenase products in the local accumulation of neutrophils in dermal inflammation in the rabbit.

Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.