H. Van Loveren

An isometric method to study respiratory smooth muscle responses in mice

An isometric method to measure the smooth muscle tone of murine tracheas in vitro was developed. Nine trachea rings from just beneath the larynx were prepared free of excess tissue with the help of a binocular microscope. These trachea parts were slipped onto supports in an organ bath containing Krebs solution. Isometric tension was measured with a force displacement transducer connected to the upper-trachea support, and is expressed as changes in grams force. The cholinergic agonist carbachol contracted isolated tracheas. Serotonin also induced contractions, but was less potent than carbachol. Histamine induced tracheal contractions only at very high concentrations. Sympathomimetic beta adrenergic agonists relaxed carbachol-precontracted tracheas with the following order of potency: isoprenaline (beta 1 and beta 2 adrenoceptor agonist) greater than salbutamol (beta 2 adrenoceptor agonist) greater than prenalterol (beta 1 adrenoceptor agonist). Adrenaline and noradrenaline relaxed carbachol-precontracted tracheas, with adrenaline being the more potent relaxant. For the study of airway reactivity, this mouse trachea model has several advantages over immunopharmacologic models: The immune system of the mouse has been characterized extensively, and many reagents are available to study the immune system and, thus, possible interactions of this system with pharmacological mechanisms. Other animal models used in pharmacology are generally less well defined immunologically.

Functional characterization of muscarinic receptors in murine airways.

1 The effects of muscarinic receptor antagonists considered to be selective for M1 receptors (pirenzepine; PZ), M2 receptors (AFDX‐116), and for M3 receptors (4‐diphenyl acetoxy N‐methylpiperidine (4‐DAMP)) were used to investigate the existence of muscarinic receptor subtypes in murine airways. Atropine was used as a nonselective antagonist. The effects of these antagonists were studied upon tracheal contractions induced either by EFS (electric field stimulation) or by application of an exogenous cholinoceptor agonist (arecoline). 2 The muscarinic receptor antagonists tested inhibited arecoline‐induced tracheal contractions with the following rank order of potency: 4‐DAMP = atropine > pirenzepine = AFDX‐116. The rank order of potency of the muscarinic antagonists used in inhibiting EFS‐induced tracheal contractions was: 4‐DAMP = atropine > PZ > AFDX‐116. The pA2 values for these antagonists were similar when compared to the pA2 values determined in guinea‐pig and bovine airway smooth muscle. 3 In addition to in vitro studies, the effects of inhalation of the different muscarinic antagonists on lung function parameters in vivo were investigated. Inhalation of 4‐DAMP induced a decrease in airway resistance and an increase in lung compliance. In contrast, inhalation of AFDX‐116 induced an increase in airway resistance and almost no change in lung compliance. Apart from some minor effects of atropine on airway resistance, atropine, PZ, and pilocarpine failed to induce changes in lung mechanics as determined by in vivo lung function measurements. 4 The results provide evidence for the existence of M3 receptors on murine tracheae that are involved in the contraction of tracheal smooth muscle. This is in agreement with other animal species such as the guinea‐pig and bovine. In vivo experiments also demonstrated that in the mouse, M3 receptors play an important role in bronchial smooth muscle contraction and thus in bronchoconstriction. Interestingly we have also demonstrated that M2 receptors can play a role in bronchodilatation. Inhalation of an M2 receptor antagonist induced an increase in airway resistance whereas inhalation of an M3 receptor antagonist induced a decrease in airway resistance. It is therefore likely that an M3/M2 receptor balance plays an important role in the regulation of airway function.

A Macrophage Factor Enhancing the Systemic Anti-Tumour Effect of T Lymphocytes

Spleen cells sensitized to tumour cells have an anti-tumour effect on injected syngeneic lymphosarcoma cells in mice. This study shows that this anti-tumour effect can be enhanced by induced peritoneal macrophages and by macrophage-like tumour cells (macrophages). Addition of macrophages to the intraperitoneally injected sensitized spleen cells stimulated the anti-tumour effect. This was observed both with intraperitoneally injected tumour cells and with subcutaneously transplanted tumour cells. The anti-tumour effect is the result of a cooperation between T cells and macrophages. In vitro incubation of immune T-cells with macrophages or macrophage-like cells enhanced the in vivo anti-tumour activity of the sensitized T-lymphocytes. Neither the presence of antigen nor the proliferation of the immune T-cells were a prerequisite to enhance this anti-tumour effect. Our experiments suggest that a macrophage factor is responsible for the enhancement of the anti-tumour effect. Based on the results of this paper and other studies we propose the following sequence of events to explain the anti-tumour effect of injected sensitized T-lymphocytes and macrophages: injected macrophages enhance the anti-tumour effect of sensitized lymphocytes. These stimulated lymphocytes migrate to the tumour located elsewhere and recognize the tumour antigens. Subsequently, the lymphocytes render (host) macrophages in the tumour cytotoxic to tumour cells.

The saccus dorsalis of the rainbow trout, Salmo gairdneri Richardson

SummaryThe saccus dorsalis of the brain of the rainbow trout, Salmo gairdneri Richardson, has been investigated by means of histological, cytochemical, enzyme-cytochemical, electron microscopical and autoradiographical techniques.The saccus dorsalis is a rostro-dorsal evagination of the diencephalic roof, and consists of a partly folded epithelial wall separating the cerebrospinal fluid from the meningeal matrix fluid. The well-developed vascular system around the epithelial wall, consisting of capillaries with different diameters, seems to be part of the pineal vascular system. No structures were found that may be involved in a possible mechanical or nervous blood flow control.The single-layered epithelium consists of highly specialized cells of one specific type. These cells are mainly characterized by infolded basal membranes, long microvilli of a peculiar shape, non-folded lateral membranes bordering intercellular spaces, apical concentrations of elongate and cupshaped macromitochondria, a basally located rough endoplasmic reticulum, an apically situated smooth endoplasmic reticulum and apical concentrations of micropinocytotic vesicles. Morphological evidence is presented of a multiple function of these cells: (1) fluid secretion, (2) extrusion of low molecular weight organic substances into the ventricular system, (3) uptake of high molecular weight substances, and (4) uptake of low molecular weight organic substances (aminergic neurotransmitters [GABA]) from the cerebrospinal fluid. The significance of light and dark cells is discussed. Indications of a possible innervation of the saccus dorsalis epithelial cells were not observed.The functional significance of the saccus dorsalis (possible analogue of the choroid plexus?) is discussed.

Macrophage‐Like Tumor Cells as Tools to Study Chemoattractive Activity

Macrophage‐like tumor cells can be obtained in large quantities as rather homogeneous populations, making these cells useful for chemotaxis assays. Therefore, macrophage‐like cells J774A, WEHI‐3, P388D1, IC‐21, and NCTC 1469, all of murine origin, and U937 of human origin, were tested for chemotactic activity to a number of chemoattractive agents, such as casein, an N‐formyl tetrapeptide (N‐formyl‐L‐norleucyl‐L‐leucyl‐L‐phenylalanyl‐L‐tyrosine), and culture supernatants of murine SL2 lymphoma cells. J774A and WEHI‐3 macrophage‐like cells of murine (BALB/c) origin expressed the strongest chemotactic activity to casein and N‐formyl tetrapeptide, respectively. The results show that: very standardized chemotaxis assays can be performed using these cell lines; these assays require appropriate cell line–stimulus combinations; there are substantial differences among cell lines as to sensitivity to various chemoattractive substances; macrophage cell lines and functional mutants may be helpful for the study of receptors for chemotaxins and the study of transducer signals for chemotaxis.

Identification and Partial Characterization of a T-Cell-Derived Antigen-Binding Factor from Mice Infected with the Intestinal Helminth Trichinella spiralis

Immunochemical and biological characterization was performed of an antigen-binding factor derived from culture supernatants of T cells from mice infected 4 days previously with the intestinal helminth Trichinella spiralis. Affinity chromatography with T. spiralis antigen resulted in the purification of a protein, provisionally designated Trichinella factor (Tric-F), that shared antigenic and other properties with a known T-cell-derived antigen-binding factor of different antigenic specificity, picryl chloride factor, which mediates an early 2-hour component of contact sensitivity. Tric-F lacked determinants of immunoglobulins and possessed determinants shared by other antigen-specific T cell factors, as determined by ELISA and antibody affinity chromatography. Biological activity of Tric-F was assayed in vivo and in vitro. Mice injected intravenously with Tric-F developed an antigen-specific early 2-hour ear swelling response following local challenge with T. spiralis antigen. These results corresponded to delayed-type hypersensitivity responses in the ears of T. spiralis-infected mice that comprised early 2-hour and late classical 24-hour responses. In vitro, Tric-F induced serotonin release by mast cells in the presence of T. spiralis antigen. Mast cells sensitized with Tric-F formed rosettes with antigen-coated sheep erythrocytes. It is suggested that Tric-F, an antigen-binding molecule that is T-cell-derived, mediates the early 2-hour component of delayed-type hypersensitivity and is involved in the initiation and regulation of T-cell-mediated intestinal inflammation during a T. spiralis infection in mice.

Enzyme-cytochemistry of the saccus dorsalis of the rainbow trout, Salmo gairdneri Richardson

SummaryIn the saccus dorsalis of the rainbow trout, Salmo gairdneri Richardson, the activity of various enzymes (transferase, lyases, oxidoreductases, hydrolases) have been studied in detail.The results of this enzyme-cytochemical study firmly demonstrate that the organ is metabolically highly active. The epithelial cells have a strong energy metabolism. Energy production can take place under aerobic as well as under anaerobic conditions. Evidence is presented that glucose from blood is directly utilized for energy demands. The epithelial cells show also high synthetic activities. The moderate amino acid metabolism may participate in the synthesis of an acid mucopolysaccharide-protein complex, especially in the so-called dark cells. Lipid metabolism appears to be restricted to the mitochondria, indicating a high turnover of lipid moieties in the membranes. In contrast to the normal looking mitochondria, the macromitochondria — besides shape and localization — have an extremely high lipid and monoamine metabolism, which may point to a special function in the cellular economy. The high activity of enzymes involved in the degradation of monoamines and in the hydration of CO2 is of particular physiological interest. The significance of the observations is discussed in relation to formerly obtained indications on the involvement of the saccus dorsalis in fluid secretion, extrusion of organic substances of low molecular weight into the ventricular system and uptake of organic substances from the cerebrospinal fluid.The hypothesis of the saccus dorsalis being an analogue of the choroid plexus is supported by several relevant data.

Antigen-Specific T-Cell Factors Induce Isotype-Like Suppression of Mast Cell and Eosinophil-Rich T-Cell-Dependent Inflammation in the Intestine of Mice Infected with Trichinella spiralis

The recent identification of a T-cell-derived antigen-binding molecule (TABM), Trichinella spiralis factor (Tric-F), isolated from culture supernatants of lymphoid cells from mice infected with the intestinal helminth T. spiralis, has led to investigation of the ability of Tric-F to induce a T-cell-dependent feedback circuit that ultimately suppresses the production of other TABMs with similar (isotype-like) features. This form of regulation that has been identified in contact hypersensitivity and in delayed-type hypersensitivity (DTH) responses to tumor cells, was shown not to be antigen-specific but to be DTH-specific. Injection of mice with the TABM called picryl chloride factor (PCl-F) induced suppression of the production of DTH-initiating TABMs of other antigenic specificities. In this study, we report that intravenous injection of mice with Tric-F or PCl-F, 8 days before an oral infection with T. spiralis, induced suppressor cells that inhibited the T-cell-dependent influx into the gut of inflammatory cells, comprising mast cells and eosinophils. Similar results were obtained when the mice were skin sensitized with PCl 8 days prior to a T. spiralis infection, i.e. in a system where TABMs are known to be produced. The phenotype of these suppressor cells was Lyt-1-2+. This suppression preferentially affected the parasite-induced DTH-like response in the gut. In contrast, increased levels of IgA plasma cells in the gut, and worm expulsion were not affected by these treatments. In reciprocal experiments, intravenous injection of Tric-F, or PCl-F, or an oral infection with T. spiralis (that results in the production of TABMs) given 8 days before contact sensitizing mice with PCl, resulted in a suppression of elicitation of cutaneous DTH, as measured by ear swelling. In contrast, pretreatment with anti-dinitrophenyl IgE antibody did not interfere with intestinal inflammation to T. spiralis nor with DTH to PCl. Our results suggest that similar to cutaneous DTH, T. spiralis-specific T-cell factors are involved in the initiation and regulation of the DTH-like mast cell and eosinophil-rich intestinal inflammation that accompanies T. spiralis infections in the gut. Since both Tric-F and PCl-F induce suppression of cellular immune responses in vivo, independent of antigen specificity, it is concluded that Tric-F belongs to the same isotype of TABMs as PCl-F that therefore can be regulated by a non-antigen-specific, isotype-like, T-cell-dependent feedback mechanism.