In vivo changes in plasma acute phase protein levels in the rat induced by slow release of IL-1, IL-6 and TNF.

Administration of large doses of cytokines by injection is required to induce changes in acute phase protein levels. Comparisons were made in the rat of the effects of administering recombinant human cytokines by injection with continuous release from implanted osmotic minipumps. Continuous release of interleukin-1beta (0.2-2.1 ng h(-1)) induced dose-related changes in the plasma levels of albumin, seromucoid proteins, haptoglobin and caeruloplasmin; interleukin-1alpha had similar effects but required higher doses (2-21 ng h(-1)). Tumour necrosis factor alpha (50 ng h(-1)) only significantly increased seromucoid levels, whereas IL-6 (3-30 ng h(-1)) induced haptoglobin and caeruloplassynthesis. This method provides a better technique for studying the in rive effects of cytokines which may be relevant to the release mechanisms in inflammation.

A novel source of mast cells: the human placenta.

The presence of moderate amounts of histamine in the human placenta was confirmed (0.72 +/- 0.10 microgram/g wet weight), and the hitherto unknown storage site of this biogenic amine was elucidated. Mast cells were identified by their characteristic morphology, staining reactions and secretory activity measured in terms of histamine release. Human placental tissue contains 7.6 x 10(5) mast cells/g wet weight, identified by staining with toluidine blue or alcian blue, and these cells were positive for chloro-acetate-esterase. Light microscope studies of placental tissue stained with HRP-conjugated anti-human IgE demonstrated cells with a typical 'halo' effect indicating cell-bound IgE, and electron microscopy revealed cells containing membrane-bound electron dense granules. A single mast cell was calculated to contain approximately 1 pg of histamine. Enzymatic digestion of placental tissue with collagenase (1.5 mg/ml) yielded viable cell suspension. containing mast cells in a purity of 0.6% which exhibited a low spontaneous output of histamine (12%). Placental mast cells released histamine in a concentration dependent manner upon challenge with anti-human IgE and the calcium ionophore A23187. Also, unlike other human mast cells so far studied. with the exception of skin, those dispersed from human placenta were responsive to the polybasic secretagogue compound 48/80. These findings represent a novel source of human mast cells and, since placentas are readily available in quantity, such tissue is proposed as an ideal source of mast cells for biochemical and pharmacological use.

Recognition of two distinct major antigens by bullous pemphigoid sera.

Sera from 17 patients with bullous pemphigoid identified a range of polypeptides of relative molecular mass (Mr) 240,000, 230,000, 190,000, 180,000, 120,000 and 100,000 from extracts of SCaBER cells, cultured human keratinocytes or human epidermis, using an immunoblotting technique. The pattern of polypeptides was characteristic for the patient serum and individual sera identified similar polypeptides from all three substrates. All 17 sera recognized major polypeptides of either Mr 230,000 (11 sera) or Mr 180,000 (seven sera) under the denaturing conditions used for immunoblotting studies. Sera from 12 patients were also examined using an immunoprecipitation technique. Polypeptide(s) of Mr 230,000 were immunoprecipitated from extracts of SCaBER cells by 11 of these sera, despite immunoblotting patterns of Mr 180,000 (or less) for three of the 11 sera. None of the minor polypeptides recognized in immunoblotting studies were immunoprecipitated by these sera. Localization of antigens was determined by binding of sera to intact or permeabilized SCaBER cells in an ELISA. Sera which recognized the Mr 230,000 polypeptide under denaturing conditions also identified an intracellular epitope in SCaBER cells, while sera which identified the denatured Mr 180,000 polypeptide bound to a cell surface epitope. Two distinct major antigens are recognized by bullous pemphigoid sera. These both appear as molecules of Mr 230,000 under non-denaturing conditions, but only one of the molecules is dissociated to produce a Mr 180,000 polypeptide under denaturing conditions. Epitopes on these two major antigens are localized on either side of the cell membrane.

Differential release of histamine and 5-hydroxytryptamine from rat mast cells: the contribution of amine uptake to the apparent pattern of secretion.

Despite the fact that mast cells isolated from rat mesentery and lung tissue contain 5-HT in excess of histamine, the pattern of amine output, in response to compound 48/80, mirrors that in peritoneal mast cells where histamine is in excess of 5-HT, such that this secretagogue induces a greater percentage release of histamine than 5-HT. Mast cells are capable of taking up both these amines, although uptake of 5-HT is in preference to that of histamine. With concentrations of clomipramine and fluoxetine that inhibited 5-HT uptake, the net percentage release of 5-HT increased in a corresponding manner, and the concentration-effect curves in response to compound 48/80 ran in a collinear fashion with that of control histamine output (measured in the absence of the drugs). Both drugs had no effect upon the uptake or secretion of histamine. Thus, observed differences in the apparent pattern of histamine and 5-HT secretion from rat mast cells may be due to selective reuptake of amines rather than a reflection of differential amine release.

The activity of amitriptyline as a differential inhibitor of amine secretion from rat peritoneal mast cells: the contribution of amine uptake.

Amitriptyline, at a concentration of 10(-4) M, significantly inhibited the release of histamine from purified peritoneal rat mast cells in response to compound 48/80, but was without effect upon the output of 5-HT. The reduction was not extensive, and concentrations of the drug above or below 10(-4) M were without effect. Amitriptyline (10(-8)-10(-4) M) inhibited uptake of exogenous 5-HT in a concentration dependent manner. Paradoxically, however, histamine uptake was significantly increased in the presence of this drug, but only at a concentration of 10(-4) M. This effect was observed at 4 degrees C for both amines. Concentrations of amitriptyline in excess of 10(-4) M caused lysis of the mast cells. These results suggest that the observed selective inhibitory effect of amitriptyline upon histamine output may be a function of altered amine uptake rather than differential inhibition and 5-HT secretion.

The protective effects of cell-free fluid exudate on cartilage degradation in vitro.

In a short-term culture system, polymorphonuclear neutrophils (PMNs) were shown to cause significant loss of glycosaminoglycan (GAG) from cartilage. In addition, a lysate of these cells, and in particular cells stimulated with a phorbol ester, greatly enhanced this loss of GAG. This loss could be partially or completely inhibited by the addition of cell-free fluid exudate to the system. In other long-term culture experiments, IL-1a was shown to enhance fibroblast-induced GAG loss from cartilage. In this system too the breakdown was inhibited by cell-free fluid exudate. Initial characterization of the protective agent or agents shows them to be heat-stable at 56 degrees C and to be non-dialysable. It is proposed that in the chronic inflammatory joint disease, rheumatoid arthritis, the fluid phase of an inflammatory response may have a protective effect against the underlying pathological processes.

The chemotactic properties of cartilage glycosaminoglycans for polymorphonuclear neutrophils.

Cartilage breakdown products have been tested for their effects on the locomotion of rat polymorphonuclear neutrophils in vitro. Chondroitin sulphate, keratan sulphate and hyaluronic acid were used in checkerboard migration assays to differentiate chemotaxis from chemokinesis. All test substances showed both chemokinetic and chemotactic activities. The same substances were then injected intradermally to determine their effects on vascular permeability and leukocyte accumulation in vivo. There was no significant effect on vascular permeability 30 minutes after injection as measured by a dye-leakage method. Histological examination of skin sections taken 6 hours after injection showed modest accumulations of polymorphonuclear neutrophils. It is suggested that cartilage breakdown products may account for the persistence of polymorphonuclear neutrophils in some chronic inflammatory joint diseases.

Comparison of histamine and 5-hydroxytryptamine content and secretion in rat mast cells isolated from different anatomical locations.

Rat mast cells, isolated from different anatomical locations, were found to exhibit heterogeneity with respect to histamine and 5-hydroxytryptamine (5-HT) content. Unlike peritoneal mast cells, those derived by enzymatic digestion of rat lung and mesentery tissues contained amounts of 5-HT in excess of histamine. Both compound 48/80 and A23187 were more effective liberators of histamine than 5-HT in each cell type, despite their differences in relative amine content. However, in response to a mixture of specific antigen and phosphatidylserine, the pattern of secretion of both amines from each mast cell type was observed to be essentially colinear. These results are discussed in terms of amine release mechanisms and post-secretory events.

Contribution of post-secretory mechanisms to the observed pattern of histamine and 5-hydroxytryptamine secretion from peritoneal rat mast cells in response to compound 48/80.

Compound 48/80 induces a greater percentage release of histamine than 5-HT from purified peritoneal rat mast cells such that the log-concentration effect curves diverge. Mast cells were shown to remove histamine and 5-HT applied exogenously, although uptake of 5-HT was in preference to that of histamine. At concentrations of clomipramine and fluoxetine that were shown to inhibit 5-HT uptake, the net percentage release of 5-HT increased in a corresponding manner. Likewise, pargyline and nialamide, which inhibit monoamine oxidase, potentiated the release of 5-HT, although they were without effect upon 5-HT uptake. Observed differences in the pattern of histamine and 5-HT secretion are proposed to be the result of post-secretory events involving amine reuptake and metabolism rather than a reflection of differential release mechanisms.

Binding of bullous pemphigoid and pemphigus vulgaris sera to SCaBER cell line in an ELISA.

Specific antibodies present in the sera of patients with bullous pemphigoid or pemphigus vulgaris were detected in an enzyme-linked immunosorbent assay (ELISA) employing a squamous carcinoma cell line, SCaBER, as substrate. Bullous pemphigoid sera bound preferentially to permeabilized cells, suggesting that the antigens are largely intracellular. The assay may prove to be a useful addition to current methods of detecting circulating antibodies in these patients.

Heparin and acute inflammation in the rat.

The subcutaneous injection of heparin into the dorsal cervical region of rats 30 min before the intrapedal administration of carrageenin significantly impaired the local inflammatory reaction from 2 h through to 6 h. This observation implies that the release of endogenous heparins from tissue mast cells may play a role in modulating the extent of the response to inflammatory insults.

Mechanism of histamine release from rat isolated peritoneal mast cells by dextran: the role of immunoglobulin E.

In vivo passive sensitization of rat peritoneal mast cells with Nippostrongylus braziliensis antiserum or rat monoclonal myeloma IgE greatly enhanced histamine release in vitro by dextran or anti IgE, but did not alter release by compound 48/80 or A23187. Conversely, removal of IgE from the cells by acid pH abolished histamine release by dextran and anti IgE but did not impair release by compound 48/80. Whereas, histamine release from cells isolated from rats genetically resistant to dextran (NR rats) by anti IgE was potentiated by passive sensitization, dextran was unable to stimulate secretion from control or sensitized NR cells. The results suggest that dextran releases histamine by interaction with cell-fixed IgE and that the NR mast cell membrane lacks the ability to interpret this stimulus.

The effects of cyclophosphamide on delayed hypersensitivity in rats.

Pretreatment of Tuck Wistar rats with cyclophosphamide (100 mg kg-1) intraperitoneally prevents the development of adjuvant-induced arthritis, experimental allergic encephalomyelitis and the delayed skin reactions after ovalbumin and Freund's incomplete adjuvant. The resistance of these rats to these stimuli is similar to that found in untreated NR Wistar rats which are resistant to dextran. However, cyclophosphamide suppresses the responses of Tuck Wistar rats to collagen-induced arthritis and sheep red blood cell agglutination, stimuli to which untreated NR Wistar rats respond. Nevertheless, the reactions of Tuck animals to the purified protein derivative of M. tuberculosis, the skin sensitisation to oxazolone, and the development of the delayed reaction after ovalbumin and Freund's complete adjuvant remain unchanged after cyclophosphamide pretreatment. The results indicate the possible existence of two separate types of delayed hypersensitivity reactions.

Susceptibility of two colonies of Wistar rats to inflammation, with particular reference to delayed hypersensitivity.

Two colonies of Wistar rats were tested for their abilities to produce delayed hypersensitivity reactions and other forms of inflammation. Tuck rats, which respond to dextran with an anaphylactoid reaction, produced delayed reactions to tuberculin protein and to ovalbumin in Freund's incomplete adjuvant. On the other hand, NELP rats which do not respond to dextran also produced delayed reactions to tuberculin protein, but only to ovalbumin when this was contained in Freund's complete adjuvant. Rats of both colonies responded to cotton pellet-induced inflammation, but the adult NELP rats showed resistance both to adjuvant-induced and to collagen-induced arthritis, as well as to the production of experimental allergic encephalomyelitis. NELP rats also showed a much greater reticulo-endothelial system phagocytic activity although antibody titres to sheep red blood cells and the mitogenic activity of concanavalin A and of lipopolysaccharide on spleen cells were similar in the two colonies of rats.

Genetic susceptibility of rats to some forms of delayed hypersensitivity.

The F1 generation, produced by mating Wistar rats from the Tuck colony which react to dextran (R rats) with Wistar rats from the NELP colony which do not react (NR rats) all responded to dextran with an anaphylactoid oedema reaction, and developed arthritis after adjuvant injections (AIA), but failed to show ovalbumin-induced delayed hypersensitivity reactions (OA) and experimental allergic encephalomyelitis (EAE). Back-crossing the F1 generation with NR rats produced animals, only half of which responded to dextran, though all the offspring reacted well to AIA, but poorly to OA and EAE. This confirms that resistance to AIA is not associated with resistance to dextran.

Distribution of 3H-dextran in two colonies of Wistar rats after intravenous injection.

The rate of elimination of 3H-dextran from the blood after intravenous administration was found to be considerably faster in rats which react to dextran with an anaphylactoid reaction than in rats which do not. Correspondingly, the rate of appearance of 3H-dextran in the anaphylactoid target organs such as the footpad was faster in reactive rats, approximately three times as much radioactivity being located in their footpads, 30 min after injection. This difference in distribution may reflect differences in the sensitivity of mast cells of the two rat colonies to dextran.

A possible link between the ability of rat isolated peritoneal mast cells to release histamine when challenged with dextran and the proteins in their plasma membranes.

The plasma membranes of peritoneal mast cells from rats specifically bred for resistance to the dextran anaphylactoid reaction contain a polypeptide of molecular weight about 74,000 daltons which is deficient in those from rats which show the reaction. It is suggested that this polypeptide modifies the dextran receptor in the resistant rats so that histamine release cannot be induced when their mast cells are challenged with dextran.

Delayed hypersensitivity reactions in rats and their response to clinical dextran.

Four colonies of rats were tested for their ability to produce adjuvant-induced arthritis, oxazolone contact hypersensitivity and the dextran anaphylactoid reaction. Tuck Wistar and Hooded Lister rats, both of which respond to dextran, showed disseminated inflammatory lesions after adjuvant and exhibited oxazolone sensitisation, regardless of the age of the animal. Spontaneously hypertensive rats, which at all ages respond to dextran, also responded to adjuvant and oxazolone but only when they were young; as they grew older, this response to these two agents diminished. Wistar NELP rats, which at all ages do not respond to dextran, responded to oxazolone; sensitivity to adjuvant, however, was maximal only in young animals. The link between the ability of rats to resist the dextran anaphylactoid reaction and their failure to respond to adjuvant with disseminated inflammatory lesions has not been confirmed.