Göran Smedegård

Acute microvascular effects of PAF-acether, as studied by intravital microscopy

Application of PAF-acether (platelet-activating factor) (final concentration 1-20 nM) to the microcirculation of the hamster cheek pouch resulted in dose-dependent vasoconstriction and an increase in macromolecular permeability, as assessed by leakage of intravascular fluorescein-labelled dextran (Mw 150 000). An increase in adhering leukocytes in venules was only seen with 20 nM PAF-acether. Animals made neutropenic by treatment with antineutrophil serum raised in rabbits showed a reduced leakage response to 20 nM PAF-acether, whereas the leakage induced with 5 nM PAF-acether was unaffected. This indicates that the increase in vascular permeability induced by PAF-acether occurs both as a presumably direct effect independent of polymorphonuclear leukocytes (PMNL), and via an indirect PMNL-dependent mechanism. Intravenous administration of PAF-acether (10 micrograms/kg body weight) to hamsters resulted in rapid and transient thrombocytopenia and leukopenia whereas 1 microgram/kg only affected the leukocytes. These potent microvascular effects of PAF-acether support its proposed role as a mediator of allergic and inflammatory reactions.

Rapid invivo metabolism of Leukotriene C3 in the monkey, Macacairus

Abstract [5,6,8,9,11,12-3H6] Leukotriene C3 (5 μCi) was injected through a catheter into the right atrium of an anesthetized male monkey. Blood samples were drawn from the aorta via a second catheter. The concentration of tritium in blood decreased from 100 nCi/ml after 5 sec to 1 nCi/ml 15 min after injection, suggesting that leukotriene C3 was rapidly eliminated from the circulation. Chromatographic analyses of radioactive material in blood collected before recirculation had occurred (15 sec after injection) demonstrated that 40% of the radioactive material had been converted into two less polar metabolites. These products had the same chromatographic properties as leukotrienes D3 and E3, respectively. The results indicate that leukotriene C3 is rapidly transformed by monkey lung in vivo . Two minutes after injection, the component corresponding to leukotriene E3 was the predominating metabolite in blood.

The microvasculature of the hamster cheek pouch as a model for studying acute immune-complex-induced inflammatory reactions (with 1 color plate).

The acute microvascular reactions induced by immune complexes were studied. Hamster cheek pouch preparations of ovalbumin (OA)-immunized animals were topically exposed to different doses of OA (0.1, 1, 10 or 100 micrograms/ml). Fluorescein-isothiocyanate-labelled dextran (FITC-dextran, molecular weight 150,000) was injected intravenously as a macromolecular tracer. The microvascular alterations observed upon exposure to OA ranged, in a dose dependent way, from a mild vasoconstriction and reversible vascular leakage from postcapillary venules exhibited by 0.1 micrograms/ml OA, to a severe long-lasting vasoconstriction, platelet aggregation, stasis, leukocyte accumulation in venules and prolonged vascular leakage from venules seen after exposure to 100 micrograms/ml OA. Exposure of immunized animals to 100 micrograms/ml fluorescein-labelled OA (FITC-OA) resulted in deposition of FITC-OA at the walls of small venules, indicating immune complex formation at these sites. A more diffuse pattern of FITC-OA localization at venules was seen with 10 micrograms/ml FITC-OA, which may possibly indicate formation of smaller more soluble immune complexes with lower doses of OA. The intravital model used in this study forms a basis for studying immunologically induced acute inflammatory reactions at the microvascular level.

Sulfasalizine aggravates experimental autoimmune encephalomyelitis and causes an increase in the number of autoreactive T cells

Sulfasalazine (SASP; 5-(p-(2-pyridylsulfamoyl)phenylazo)salicyclic acid) has beneficial effects on certain inflammatory diseases and has been proposed for clinical trials in multiple sclerosis (MS). We have explored the effects of SASP on actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. SASP was given orally at three different doses from the day of immunization to day 40 post-immunization (p.i.). All doses led to a clinically more protracted disease, increased numbers of T cells infiltrating into the central nervous system (CNS) and to increased numbers of interferon-gamma-secreting cells (IFN-gamma-sc) in the CNS. The effects of SASP treatment on T cell-mediated autoimmunity against CNS myelin and peptides of myelin basic protein (MBP) were measured by IFN-gamma secretion and proliferation by lymph node mononuclear cells in response to these antigens. In SASP-treated rats, increased numbers of IFN-gamma-sc appeared in response to myelin antigens, while the proliferative responses were decreased. We suggest that monitoring cell-mediated immunity with the IFN-gamma-sc method may be relevant for the evaluation of new immunotherapeutic strategies in inflammatory demyelinating diseases. Furthermore, our results demand caution as to clinical trials with SASP in MS.

Aggregate Anaphylaxis in the Monkey

Haematological and histological studies were made following challenge in 8 monkeys (Macaca irus) sensitized with ovalbumin. Haemagglutinating, but no reaginic antibodies to ovalbumi

Immune-complex-induced inflammatory reaction studied by intravital microscopy Role of histamine and arachidonic acid metabolites

An immune-complex-induced inflammatory reaction was elicited in the hamster cheek pouch microvasculature of ovalburnin (OA)-immunized animals by exposure to 1 or 100 μg/ml OA. The low antigen dose caused arteriolar constriction, transient platelet aggregation, and a reversible increase in vascular leakage at postcapillary venules. With the high antigen dose, the constriction and platelet aggregation were more pronounced and the vascular leakage was prolonged. This antigen dose also caused a massive PMNL accumulation in small venules, which coincided with the prolonged vascular leakage. Histamine was released in the reaction as pretreatment with mepyramine largely inhibited the leakage response to 1 μg/ ml OA. With 100 mg/ml OA, only the initial phase of vascular leakage was inhibited by mepyramine, leaving the prolonged vascular leakage and PMNL accumulation unaltered. Pretreatment with methylprednisolone 16–18 h prior to the experiments reduced both phases of vascular leakage as well as the PMNL accumulation. Pre-treatment with the combined cyclo- and lipoxygenase inhibitors BW755C or nordihydroguaiaretic acid (NDGA) reduced the initial vasoconstriction induced with 100 μg/ml OA, thereby augmenting the initial vascular leakage. Despite this, the prolonged phase of vascular leakage was reduced in the NDGA-treated animals. Cyclooxygenase products were not found to play a crucial role in mediating the vascular response; on the contrary, indomethacin pretreatment slightly potentiated the vascular leakage induced by the low antigen dose.

Anaphylaxis in the Monkey: Respiratory Mechanics, Acid‐base Status and Blood Gases

Aggregate anaphylaxis was induced by intravenous injection of the specific antigen in eight ovalbumin‐sensitized monkeys. Changes in respiratory mechanics, acid‐base status and blood gases were studied during the following half hour. Within 1 minute after challenge, a short period of respiratory depression, probably reflex‐mediated, was observed. This was followed by hyperventilation, and arterial Pco2 decreased. There was a rapid increase in pulmonary resistance (Rpulm) and a concomitant decrease in pulmonary dynamic compliance (Cdyn), suggesting constriction of smooth muscles in the lung. Rpulm returned to the control value but Cdyn remained depressed, as a result of constriction of small airways and pulmonary congestion. Oxygen saturation in arterial blood decreased slightly due to a marked destruction of mixed venous blood and increased venous admixture. Progressive metabolic acidosis developed, indicating poor tissue oxygenation and perfusion. The changes observed in this study were not severe enough to cause any major disturbance of the gas exchange in the lungs, despite a severe anaphylactic shock.

Permeability-increasing ability of PAF-acether in rat skin

Platelet-activating factor (PAF-acether), a phospholipid compound with effects on several cells, e.g., platelets and polymorphonuclear leukocytes (PMNs), was examined for its effect on microvascular permeability in rat skin. It was found to increase microvascular permeability, measured as exudation of [125I]human serum albumin, in amounts exceeding 1 pmol, and was more than 1000 times as potent as histamine. The effect was independent of cell infiltration, as no accumulation of PMNs, measured as the amount of myeloperoxidase in the skin, occurred and as the response was unaltered in animals rendered neutropenic due to treatment with an antiserum against PMNs.

Histopathological lung changes in immune complex mediated anaphylactic shock in humans elicited by dextran

Severe dextran-induced anaphylactic reaction (DIAR) is being recognized as a form of immediate IgG mediated immune complex reaction. Support for this pathogenesis is found in the correlation between the titer of dextran-reactive antibodies of IgG class and the severity of the reaction. Autopsy records were reviewed in 27 certified cases of fatal DIAR. The most frequent macroscopic findings were dilatation of the right side of the heart and acute pulmonary stasis. Autopsy lung specimens were collected from 17 of these patients. In 15 of the 17 lung specimens pulmonary microemboli were found. The microemboli had the appearance of hyaline eosinophilic globules, and the lung vasculature also contained leukocytes, platelets and disintegrated erythrocytes. These findings show similarity to the findings in a monkey model of known IgG mediated anaphylaxis, and give further support to the proposed pathogenesis of severe DIAR.

Anaphylaxis in the Monkey: Pulmonary Oedema after Pre-treatment with β-Receptor Stimulants

Aggregate anaphylaxis was induced in seven ovalbumin‐sensitized monkeys, with high titres of ovalbumin specific haemagglutinating antibodies. After pre‐treatment with an intravenous (i.v.) injection of 0.25 mg/kg terbutaline (n = 6) cr an infusion of isoprenaline (n = 1), anaphylactic shock was induced by i.v. challenge with specific antigen. Haemodynamics, regional blood flows, respiratory mechanics, blood gases and haematological changes were studied during the following 30 min. Severe shock developed following ovalbumin challenge and the cardiac output was reduced by a mean of 74%. Pulmonary vascular resistance increased 11‐fold. Pulmonary dynamic compliance decreased, but there was only a minor increase in pulmonary resistance. Hypoxaemia and severe metabolic acidosis developed. Circulating platelets and leucocytes decreased markedly. Three animals died with fulminant pulmonary oedema. In conclusion, the reaction pattern was similar to that found in studies of monkeys that received no prior treatment. However, the occurrence of pulmonary oedema suggests that the effects of large doses of terbutaline on the heart, combined with the high pulmonary vascular resistance, resulted in more severe pulmonary changes than took place in untreated animals.